( B ) CTB - labeled dLGN shows reduced eye - segregation at P30 in CKO mice . The left column shows contralateral dLGN labeled with CTB488 ( green ) , and the middle one is ipsilateral dLGN with CTB647 ( magenta ) . The right column represents the dLGN pseudocolored according to the R - value for each pixel ( R = log ( Fipsi / Fcontra ) ) . ( C ) A histogram distribution chart of R - value for all pixels within dLGN represents the degree of eye - specific segregation . When R is 0 , it means equal ipsilateral and contralateral fluorescence intensity at a pixel . A greater R - value means a bigger difference of ipsi - to - contraleteral fluorescence intensity . The narrower distribution of CKO in the inset indicates reduced segregation . ( D ) The variance of R distributions in P30 control and CKO mice . N = 4 ( Ctrl ) , N = 6 ( CKO ) , p = 0 . 004 , mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CTB-labeled dLGN shows reduced eye-segregation at P30 in CKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/Fcontra)). (C) A histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. When R is 0, it means equal ipsilateral and contralateral fluorescence intensity at a pixel. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of CKO in the inset indicates reduced segregation. (D) The variance of R distributions in P30 control and CKO mice. N = 4 (Ctrl), N = 6 (CKO), p = 0.004, mean ± SD.


( F ) Maximal NMDAR mediated currents measured in the dLGN of P28 ~ P34 mice , p = 0 . 03 , N = 14 ( Ctrl ) , 23 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

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(F) Maximal NMDAR mediated currents measured in the dLGN of P28~P34 mice, p = 0.03, N = 14 (Ctrl), 23 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( G ) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice . ( H ) Quantification of NMDAR1 expression in CKO mice and control .

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(G) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice. (H) Quantification of NMDAR1 expression in CKO mice and control.


( I ) Maximal AMPAR mediated currents measured in the dLGN of P28 ~ P34 mice . P = 0 . 302 , N = 13 ( Ctrl ) , 17 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

(I) Maximal AMPAR mediated currents measured in the dLGN of P28~P34 mice. P=0.302, N = 13 (Ctrl), 17 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( J ) Western blot of GluR1 with microdissected P30 dLGN tissue . ( K ) Quantification of GluR1 expression in CKO mice and controls .

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(J) Western blot of GluR1 with microdissected P30 dLGN tissue. (K) Quantification of GluR1 expression in CKO mice and controls.


( A ) Colocalized RGC inputs and PSVue signals in P6 control and CKO . White circles indicate PS + RGC inputs . Arrows pointing to the RGC inputs enlarged in the insets . ( B ) The percentage of PS + RGC inputs in total inputs in CKO and controls .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.


( C ) Representative images of engulfed PS + and PS - RGC inputs by microglia in control and CKO . ( D ) Quantification of engulfed PS + RGC inputs by microglia , which is calculated as : Volume of engulfed PS + RGC inputs / Volume of microglia cell . ( E ) Quantification of engulfed PS - RGC inputs in control and CKO .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO.


( a - d ) Formation of protein inclusions in SY5Y cells expressing FL ( a ) or the indicated truncated forms of Sph1 ( b - d ) was induced by 12 h lactacystin treatment . After extensive washes , cells were maintained in serum - supplemented ( serum + ) or serum - free ( serum − ) media , and in the absence ( Control ) or presence of 10 mM 3 - MA , an inhibitor of macroautophagy . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( left ) or Agg ( right ) observed under different recovery conditions . Values are mean + s . e . ( n = 4 ) . * Significant versus serum + condition . See also Supplementary Figs S1 - S5 .

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

(a-d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b-d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1-S5.


( a - c ) Top : fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL ( a ) , or the indicated truncated Sph1 proteins ( b - c ) treated with lactacystin ( 12 h ) and recovered in basal conditions ( serum + ) . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( middle ) or Agg ( bottom ) . Values are mean + s . e . ( n = 3 ) . * Significant versus control .

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

(a-c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b-c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control.


( d - f ) Relative fold difference in the number of FL ( d ) or truncated ( e - f ) Sph1 Agm ( left ) and Agg ( right ) in WT and Atg5 KO mice MEFs incubated in serum + or serum − conditions . Values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition .

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

(d-f) Relative fold difference in the number of FL (d) or truncated (e-f) Sph1 Agm (left) and Agg (right) in WT and Atg5 KO mice MEFs incubated in serum+ or serum− conditions. Values are mean+s.e. (n=3). *Significant versus serum+ condition.


( a - d ) Left : fluorescence images of SY5Y cells co - expressing HA - p38 ( red ) and GFP ( green ) forms of FL ( a ) or the indicated truncated Sph1 ( b - d ) . Cells were treated with lactacystin ( 12 h ) and recovered in the presence or absence of serum . Graphs : percentage of p38 existing alone in Agm or co - aggregating with Sph1 in the forms of Agm or Agg ( left ) , and susceptibility of p38 existing on its own in Agm or co - aggregating with Sph1 proteins in Agm toward serum - stimulated autophagic clearance ( right ) . Values are expressed as folds Agm in the presence of serum and are mean + s . e . ( n = 3 ) . * Significant versus serum + condition calculated using Student ' s t - test .

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(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test.


( e ) Autophagic susceptibility of Agm formed by p38 and ANK1 - p38 fusion proteins . Values are mean + s . e . ( n = 3 ) . * Significant versus control group calculated using Bonferroni test . See also Supplementary Fig . S6 .

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(e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.


( a ) Top : colocalization of LAMP1 ( red ) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti - GFP ( green ) in SY5Y cells . Arrows indicate colocalization . Bottom : percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum + or serum − media , treated or not with lysosomal inhibitors ( N / L ) .

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

(a) Top: colocalization of LAMP1 (red) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti-GFP (green) in SY5Y cells. Arrows indicate colocalization. Bottom: percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum+ or serum− media, treated or not with lysosomal inhibitors (N/L).


( b ) Percentage LAMP1 colocalization with Agm in cells co - expressing HA - p38 and the indicated Sph1 proteins maintained in the absence of serum .

(b) Percentage LAMP1 colocalization with Agm in cells co-expressing HA-p38 and the indicated Sph1 proteins maintained in the absence of serum.


( c ) Left : colocalization of LAMP1 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Right : percentage LAMP1 colocalization with p38 and ANK1 - p38 Agm in cells maintained in the presence or absence of serum .

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(c) Left: colocalization of LAMP1 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Right: percentage LAMP1 colocalization with p38 and ANK1-p38 Agm in cells maintained in the presence or absence of serum.


( d , e ) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP - tagged Sph1 truncations ( d ) , or p38 and ANK1 - p38 ( e ) . Top : phase - contrast fluorescence microscopy ; middle : electron micrographs of the boxed region ; bottom : higher magnification examples of autophagic vacuoles ( yellow arrows ) . # Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs .

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(d,e) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP-tagged Sph1 truncations (d), or p38 and ANK1-p38 (e). Top: phase-contrast fluorescence microscopy; middle: electron micrographs of the boxed region; bottom: higher magnification examples of autophagic vacuoles (yellow arrows). #Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs.


( f ) Top : colocalization of LC3 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Bottom : percentage colocalization of LC3 with the fluorescent proteins . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , c , f ) or p38 Agm ( b ) calculated using Bonferroni test . See also Supplementary Fig . S7 .

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

(f) Top: colocalization of LC3 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Bottom: percentage colocalization of LC3 with the fluorescent proteins. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,c,f) or p38 Agm (b) calculated using Bonferroni test. See also Supplementary Fig. S7.


( a ) Fluorescence images showing colocalization patterns of the indicated GFP - p38 variants and Sph1 ( green ) with endogenous p62 ( red ) in SY5Y cells . Arrows indicate colocalization .

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(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization.


( b ) Quantitative FRAP analysis of mCherry - p62 expressed alone ( control ) or co - expressed with the indicated GFP - Sph1 and - p38 variants . Top : time course of changes in relative fluorescence intensity ( RFI ) ; bottom : percentage of p62 in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Representative images are shown in Supplementary Fig . S8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8.


( c - d ) Top : IBs for p62 and actin in control and p62 knockdown ( p62 KD # 325 or # 788 ) SY5Y cells ( c ) ; and in control and p62 KO MEFs ( d ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells in response to serum removal .

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

(c-d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal.


( e ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1 . Arrows indicate colocalization .

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(e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization.


( f , g ) Top : IBs for NBR1 , p62 and actin in control and NBR1 knockdown ( NBR1 KD # 161 ) SY5Y cells ( f ) , and in control and p62 / NBR1 double knockdown ( Double KD # A or # B ) SY5Y cells ( g ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells . For all densitometric analyses , values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition . See also Supplementary Fig . S8 - S11 .

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(f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8-S11.


( a - d ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin - 1 ( a ) , Vps34 ( b ) , Atg14L ( c ) and DFCP - 1 ( d ) . Arrows indicate colocalization .

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(a-d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.


( a ) Quantitative FRAP analysis of the indicated GFP - p38 variants . Top : representative images . Middle : time course of changes in relative fluorescence intensity ( RFI ) . Bottom : percentage of proteins in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Scale bars , 10

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

(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm


( b ) Top : schematic showing all lysine ( K ) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site ( s ) . Bottom : relative fold difference in the number of Agm formed by WT and the different ' K to R ' mutants of ANK1 - p38 fusion protein in SY5Y cells maintained in the presence or absence of serum .

(b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different 'K to R' mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum.


( c ) IBs for HA ( top ) and GFP ( bottom ) of total lysates ( Input ) and GFP immunoprecipitates ( IP ) from the cell lysates of SY5Y cells co - transfected with the indicated HA - ubiquitin constructs and GFP - p38 or - ANK1 - p38 WT or mutant K385R in the presence of lactacystin . ( d ) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination . Values were corrected by levels of GFP immunoprecipitated .

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

(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.


( e ) Top : IF for GFP and K63 - linked ubiquitin ( red ) of SY5Y cells expressing GFP - p38 or - ANK1 - p38 WT and mutant K385R ( green ) . Arrows indicate colocalization . Bottom : percentage of Agm positive for K63 - linked ubiquitin .

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(e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin.


( f ) Quantitative FRAP analysis of GFP - ANK1 - p38 K385R and GFP - ANK1 - p38 as in ( a ) . Values are mean + s . e . of ten cells analysed over three different experiments .

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(f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments.


( g ) Top : representative images of GFP - ANK1 - p38 K385R Agm in SY5Y cells immunostained for Atg14L , Vps34 and DFCP - 1 . Bottom : percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L , Vps34 and DFCP - 1 under serum − conditions . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition or p38 Agm . # Significant versus ANK1 - p38 Agm . Significance calculated using Bonferroni test . See also Supplementary Fig . S8 .

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(g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.


( a , b ) Autophagic clearance of Agm ( a ) or Agg ( b ) formed by FL WT and mutant K385R or K394R GFP - Sph1 in SY5Y cells , in response to serum removal ( a ) or upon blockage of basal autophagy with 3 - MA ( b ) .

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

(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).


( c , d ) Left : Agm ( c ) or Agg ( d ) of FL GFP - Sph1 ( green ) immunostained for endogenous K63 - linked ubiquitin ( red ) in SY5Y cells . Arrows in ( c ) indicate colocalization . Right : percentage of Agm ( c ) or Agg ( d ) positive for K63 - linked ubiquitin .

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

(c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin.


( e ) Formation of WT and mutant K385R FL Sph1 ( left ) and ANK1 - p38 ( right ) Agm in response to proteasome inhibition . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , b ) or WT FL Sph1 or ANK1 - p38 ( c - e ) .

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

(e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c-e).


E ) Caco - 2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h . Western blotting of cell lysates is shown .

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E) Caco-2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h. Western blotting of cell lysates is shown.


D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h . D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with mouse ( m ) IFITM plasmids or vector control for 24 h . C ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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

HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


D ) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in ( C ) and one experiment with MOI 0 . 1 . Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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

D) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in (C) and one experiment with MOI 0.1. Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu - 3 cells transfected with mCherry and SARS - CoV - 2 Spike ( S ) or vector control . Cells were co - cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye . A ) Representative example fluorescent imaging of the indicated co - cultures showing the presence of EGFP / mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs . Scale bar indicates 50 μm . B ) Quantification of nuclei per EGFP / mCherry double positive cell syncytia in co - cultures involving the indicated IFITM3 constructs as compared to vector control . Bars depict averages of three independent experiments with circles representing the individual data points . For each condition , nuclei within 50 - 75 individual syncytia were counted . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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

U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h . B ) Graph depicts normalized infection percentage measurements from 3 independent experiments , each performed in triplicate , from cells infected as in ( B ) . Bars represent averages with individual data points shown as circles . Error bars represent SD . The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection . # p < 0 . 05 by ANOVA followed by Tukey ' s multiple comparisons test . NS , not significant . Not indicated , additional comparisons of interest with p < 0 . 05 include IFITM3 ( - ) vs . IFITM3 - Y20 ( - ) , IFITM3 ∆ 59 - 68 ( - ) , and IFITM3 - S61A , N64A , T65A ( - ) .

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

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h. B) Graph depicts normalized infection percentage measurements from 3 independent experiments, each performed in triplicate, from cells infected as in (B). Bars represent averages with individual data points shown as circles. Error bars represent SD. The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test. NS, not significant. Not indicated, additional comparisons of interest with p<0.05 include IFITM3 (-) vs. IFITM3-Y20 (-), IFITM3∆59-68 (-), and IFITM3-S61A,N64A,T65A (-).


( C - D ) Diverse outcome for tumor cells following interactions with CTLs . OT - I CTLs and OVA + Eµ - myc B cells were labeled with the calcium - sensitive dye Indo - 1 , cultured together in the presence of propidium iodide ( PI ) and subjected to live imaging . Representative time - lapse images ( C ) and corresponding fluorescence quantification ( D ) showing the three distinct outcomes for CTL - tumor cell contacts ( left , calcium response in T cells , and right , calcium and PI signal in targets ) : no detectable damage ( no calcium signal in the target cells ) , reversible damage ( transient calcium elevation and low uptake of PI ) , and irreversible damage ( prolonged calcium elevation and bright PI staining ) . CTLs are outlined by white dashed lines . White arrowheads indicate tumor cells . Red arrowheads indicate tumor cells with internalized PI . Scale bar , 5 µm .

O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Diverse outcome for tumor cells following interactions with CTLs. OT-I CTLs and OVA+ Eµ-myc B cells were labeled with the calcium-sensitive dye Indo-1, cultured together in the presence of propidium iodide (PI) and subjected to live imaging. Representative time-lapse images (C) and corresponding fluorescence quantification (D) showing the three distinct outcomes for CTL-tumor cell contacts (left, calcium response in T cells, and right, calcium and PI signal in targets): no detectable damage (no calcium signal in the target cells), reversible damage (transient calcium elevation and low uptake of PI), and irreversible damage (prolonged calcium elevation and bright PI staining). CTLs are outlined by white dashed lines. White arrowheads indicate tumor cells. Red arrowheads indicate tumor cells with internalized PI. Scale bar, 5 µm.


( A - B ) Calcium responses were recorded over time in ( A ) unconjugated tumor cells or in ( B ) tumor cells forming a conjugate with a CTL ( during the duration of the contacts ) . Each line corresponds to an individual tumor cell and each square to a time point . Squares are colored in magenta upon calcium elevation or in blue otherwise . Apoptotic events ( detected by caspase 3 activity ) are indicated by an overlaid green dot . Data is representative of three independent experiment .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CTL (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.


( A ) Representative time - lapse images showing tumors without CTLs ( upper panels ) or with OT - I CTLs ( lower panels ) . Tumor cells with low and high intracellular calcium appear in blue and red , respectively . CTLs appear in white . Red arrows indicate tumor cells with elevated calcium . Scale bar , 5 µm . Data information : Data were pooled of four independent experiments . ( * p < 0 . 05 , * * * p < 0 . 001 , unpaired t ‑ test ) .

O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative time-lapse images showing tumors without CTLs (upper panels) or with OT-I CTLs (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CTLs appear in white. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm. Data information: Data were pooled of four independent experiments. (*p<0.05, ***p<0.001, unpaired t‑test).


CTLs exhibit extensive differences in lytic activity in vivo . Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct - GFP - expressing OT - I CTLs . Intravital imaging of the bone marrow was performed two days after T cell transfer . ( E ) Representative two - photon time - lapse images showing a representative CTL with high lytic capacity ( killing 4 targets ) and a CTL with no lytic activity for up to 4 hours . Blue arrows mark apoptotic tumor cells . Live and apoptotic tumors appear in grey and blue , respectively . CTLs appear in green . Scale bar , 10 µm . Representative of 3 independent experiments .

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

CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (E) Representative two-photon time-lapse images showing a representative CTL with high lytic capacity (killing 4 targets) and a CTL with no lytic activity for up to 4 hours. Blue arrows mark apoptotic tumor cells. Live and apoptotic tumors appear in grey and blue, respectively. CTLs appear in green. Scale bar, 10 µm. Representative of 3 independent experiments.


( A - B ) Calcium responses were recorded over time in ( A ) unconjugated tumor cells or in ( B ) tumor cells forming a conjugate with a CAR T cell . Each line corresponds to an individual tumor cell and each square to a time point . Squares are colored in magenta upon calcium elevation or in blue otherwise . Apoptotic events ( detected by caspase 3 activity ) are indicated by an overlaid green dot . Data is representative of three independent experiment .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CAR T cell. Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.


( A ) Representative time - lapse images showing tumors without CAR T cells ( upper panels ) or with CAR T Cells ( lower panels ) . Tumor cells with low and high intracellular calcium appear in blue and red , respectively . CAR T cells appear in light green . Red arrows indicate tumor cells with elevated calcium . Scale bar , 5 µm .

O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(A) Representative time-lapse images showing tumors without CAR T cells (upper panels) or with CAR T Cells (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CAR T cells appear in light green. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm.


( B ) A fragment encompassing amino acids 432 - 520 of Atg13 ( wt ) or the corresponding FV mutant ( FV ) was expressed in E . coli and incubated in excess with an immobilized GST ‐ tagged fragment of Atg1 ( amino acids 501 - 897 ) purified from E . coli ( GST ‐ Atg1 ) . Input ( 5 . 5 % ) and bound proteins were analysed by coomassie blue staining , which stains proteins proportional to their size . The size of marker proteins ( in kDa ) is indicated on the left .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A fragment encompassing amino acids 432-520 of Atg13 (wt) or the corresponding FV mutant (FV) was expressed in E. coli and incubated in excess with an immobilized GST‐tagged fragment of Atg1 (amino acids 501-897) purified from E. coli (GST‐Atg1). Input (5.5%) and bound proteins were analysed by coomassie blue staining, which stains proteins proportional to their size. The size of marker proteins (in kDa) is indicated on the left.


( C ) ATG1 ‐ TAP atg13Δ cells containing endogenously GFP ‐ tagged Atg17 , Atg29 or Vac8 and wild ‐ type ( wt ) or the Atg13 FV ‐ mutant ( F468A ; V469A ) were grown to mid log phase . Atg1 was immunoprecipitated and its association with Atg13 and the GFP ‐ tagged proteins was analysed by immunoblotting . Extract inputs are shown in Supplementary Figure S1B .

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

(C) ATG1‐TAP atg13Δ cells containing endogenously GFP‐tagged Atg17, Atg29 or Vac8 and wild‐type (wt) or the Atg13 FV‐mutant (F468A; V469A) were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 and the GFP‐tagged proteins was analysed by immunoblotting. Extract inputs are shown in Supplementary Figure S1B.


( D ) atg1Δ and atg1Δatg13Δ cells expressing HA ‐ tagged Atg1 and wild ‐ type ( wt ) cells containing an empty plasmid were grown to mid log phase ( rich ) , and then treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting . Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS - PAGE , and the extent corresponds to the shift typically observed under our gel conditions .

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

(D) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting. Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS-PAGE, and the extent corresponds to the shift typically observed under our gel conditions.


( E ) Yeast cells containing endogenously tagged Atg1 ‐ TAP with and without endogenously tagged Atg13 ‐ GFP , or conversely cells containing endogenously tagged Atg13 ‐ TAP and Atg1 ‐ GFP expressed from a centromeric plasmid were grown to mid log phase ( rich ) and treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 ‐ TAP and Atg13 ‐ TAP were affinity purified and their association with GFP ‐ tagged proteins was analysed by western blotting .

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

(E) Yeast cells containing endogenously tagged Atg1‐TAP with and without endogenously tagged Atg13‐GFP, or conversely cells containing endogenously tagged Atg13‐TAP and Atg1‐GFP expressed from a centromeric plasmid were grown to mid log phase (rich) and treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1‐TAP and Atg13‐TAP were affinity purified and their association with GFP‐tagged proteins was analysed by western blotting.


( F ) Histone3 ‐ HA tagged Atg1 was expressed together with wild ‐ type ( wt ) or the Atg13 ‐ FV mutant fused to Suv methylase ( Suv ) in atg1Δatg13Δ cells , or for control together with Pbs2 ‐ Suv in atg1Δ cells . Logarithmically growing cultures were treated with rapamycin ( rapa ) for 0 or 120 min , and methylation was monitored by preparing cell extracts and western blotting with an anti ‐ trimethylation ‐ specific antibody . Note that Suv ‐ , GFP ‐ or TAP ‐ tagged Atg13 are fully functional , however , do not show the characteristic phosphorylation ‐ induced reduced mobility observed with the untagged protein under our gel conditions .

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

(F) Histone3‐HA tagged Atg1 was expressed together with wild‐type (wt) or the Atg13‐FV mutant fused to Suv methylase (Suv) in atg1Δatg13Δ cells, or for control together with Pbs2‐Suv in atg1Δ cells. Logarithmically growing cultures were treated with rapamycin (rapa) for 0 or 120 min, and methylation was monitored by preparing cell extracts and western blotting with an anti‐trimethylation‐specific antibody. Note that Suv‐, GFP‐ or TAP‐tagged Atg13 are fully functional, however, do not show the characteristic phosphorylation‐induced reduced mobility observed with the untagged protein under our gel conditions.


( G ) atg1Δatg13Δ cells containing TAP ‐ tagged wild ‐ type Atg1 ( wt ) or the Atg1 kinase ‐ dead ( kd ) K54A mutant and wild ‐ type Atg13 , the Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase . Atg1 was immunoprecipitated and its autophosphorylation activity was measured by autoradiography in vitro .

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

(G) atg1Δatg13Δ cells containing TAP‐tagged wild‐type Atg1 (wt) or the Atg1 kinase‐dead (kd) K54A mutant and wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase. Atg1 was immunoprecipitated and its autophosphorylation activity was measured by autoradiography in vitro.


( H ) pho8Δ60pho13Δatg13Δ cells expressing wild ‐ type Atg13 ( wt ) , the Atg13 ‐ FV mutant ( FV ) or an empty plasmid were grown to mid log phase and starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials and methods ' , and plotted as relative ALP activity with standard deviation ( s . d . ) compared to the wild ‐ type controls .

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

(H) pho8Δ60pho13Δatg13Δ cells expressing wild‐type Atg13 (wt), the Atg13‐FV mutant (FV) or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( I ) atg13 cells expressing GFP ‐ Atg8 and containing wild ‐ type Atg13 , the Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD ‐ N . Processing of endogenous Ape1 and cleavage of GFP ‐ Atg8 was analysed by western blotting , and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP ‐ Atg8 , respectively . The asterisk marks a non ‐ specific band detected by the Atg13 antibody . Figure source data can be found with the Supplementary data .

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

(I) atg13 cells expressing GFP‐Atg8 and containing wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD‐N. Processing of endogenous Ape1 and cleavage of GFP‐Atg8 was analysed by western blotting, and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP‐Atg8, respectively. The asterisk marks a non‐specific band detected by the Atg13 antibody. Figure source data can be found with the Supplementary data.


( B ) GST ‐ Atg8 or GST ‐ Ubiquitin ( Ub ) was purified from E . coli and bound to GSH beads . Wild ‐ type Atg1 ‐ TAP or the VE and EYE mutants were purified from yeast , incubated with the immobilized GST proteins and bound proteins analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting.


( C ) ( C ) GST ‐ Atg8 and GST ‐ Ubiquitin were immobilized as described in ( B ) , and probed for their ability to bind Atg1 ‐ TAP isolated from wild ‐ type or atg13Δ cells .

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

(C) (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells.


( D ) atg1Δatg13Δ cells expressing as indicated wild ‐ type Atg1 ‐ TAP or the VE or EYE mutants , and wild ‐ type Atg13 or the Atg13 ‐ FV mutant were grown to mid log phase . Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting .

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

(D) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting.


( E ) GST , GST ‐ LC3B , GST ‐ GATE16 and GST ‐ GABARAP were purified from E . coli and bound to beads . HEK293 cell lysates prepared from cells expressing wild ‐ type ( wt ) HA ‐ ULK1 or the DFP mutant were incubated with the beads , and bound proteins analysed by western blotting .

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

(E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting.


( F ) HEK293 cells were co ‐ transfected with GFP or GFP ‐ GABARAP along with either wild ‐ type HA ‐ ULK1 or the DFP mutant . GFP and GFP ‐ GABARAP were immobilized on GFP ‐ Trap resin and HA ‐ ULK1 binding was analysed by western blotting . Figure source data can be found with the Supplementary data .

(F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.


( A ) atg1Δatg13Δ cells containing an empty control plasmid , wild ‐ type or the indicated Atg1 mutants , and Atg13 wild ‐ type , the Atg13 ‐ FVmutant or an empty plasmid were grown to mid log phase . Processing of endogenous Ape1 was analysed by western blotting , and quantified as described in the legend of Figure 1I . Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13 . The asterisk marks a non ‐ specific band detected by the Atg13 antibody .

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

(A) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FVmutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody.


( B ) pho8Δ60pho13Δatg1Δ cells expressing wild ‐ type ( wt ) , kinase ‐ dead ( kd ) , Atg1 ‐ VE ( VE ) or ‐ EYE ( EYE ) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials and methods ' , and plotted as relative ALP activity with standard deviation ( s . d . ) compared to the wild ‐ type controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( C ) Cells as in ( A ) expressing GFP ‐ Atg8 were grown to mid log phase with starvation for 4 h in SD ‐ N medium . The processing of GFP ‐ Atg8 was analysed and quantified as in Figure 1I .

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(C) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I.


( D ) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1 ‐ RFP and either GFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were examined by fluorescence microscopy . Bar = 5 μm .

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(D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm.


( E ) atg1Δ cells containing TAP ‐ tagged wild ‐ type or the indicated Atg1 ‐ mutants were grown to mid log phase . Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography . The kinase ‐ dead ( kd ) Atg1 ‐ mutant K54A serves as a negative control . Figure source data can be found with the Supplementary data .

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

(E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.


( A ) Exponentially growing ypt7Δ cells were starved in SD ‐ N medium for 4 h , lysed and the extract separated into a cytoplasmic ( S ) and a 5000 g membrane pellet fraction . The pellet was treated with ( + ) or without ( − ) TX ‐ 100 , centrifuged and the supernatant ( S ) and pellet ( P ) fractions analysed by western blotting with anti ‐ Atg1 , anti ‐ Pep12 and anti ‐ Pgk1 antibodies .

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

(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies.


( B ) Exponentially growing ypt7Δatg1Δ cells expressing wild ‐ type Atg1 or the Atg1 ‐ VE mutant protein were starved in SD ‐ N for 4 h , lysed and the extract separated into cytoplasmic ( S ) and membrane ( P ) fractions . The fractions were analysed by western blotting with anti ‐ Atg1 , anti ‐ Pgk1 and anti ‐ Ape1 antibodies , and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild ‐ type cells .

(B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells.


( C ) HEK293 cells were co ‐ transfected with GFP ‐ GABARAP and HA ‐ ULK1 , starved for 2 h , and subsequently immunostained for HA ( red ) and WIPI2 ( white ) . The arrows mark putative autophagosomes that stain positive for all three markers . Bar = 10 μm .

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(C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm.


( D ) HEK293 cells stably expressing GFP ‐ LC3 were transfected with wild ‐ type HA ‐ ULK1 or the DFP mutant , and immunostained for HA and WIPI2 after 2 h starvation . HA ‐ ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells ( from one out of two experiments ) and plotted as the ratio of ULK1 to WIPI2 puncta . * * * Indicates a statistically significant P ‐ value of 0 . 0001 . One of the two independent experiments is shown .

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(D) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown.


( E ) Quantification of HA ‐ ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild ‐ type HA ‐ ULK1 or the DFP mutant . Cells were treated as above , and the spot number counted using Imaris software . A representative immunofluorescence image is shown in Supplementary Figure S3F . * * * and * indicate P ‐ values of 0 . 0001 or 0 . 0158 , respectively . Figure source data can be found with the Supplementary data .

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

(E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.


( A ) atg1Δ cells containing either wild ‐ type Atg1 or the kinase ‐ dead Atg1 ‐ T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD ‐ N . Samples were taken at indicated time points and GFP cleavage was analysed by western blotting . Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux .

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

(A) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux.


( B ) atg1Δ cells containing either GFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown and analysed as in ( A ) .

O O O O O O 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) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A).


( C ) atg1Δpep4Δ cells containing YFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown to log phase and starved in SD ‐ N . Cells were monitored by time ‐ lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in ' Materials and methods ' . Data are plotted as the mean ± s . e . m . from at least 90 quantified cells . Additional images are shown in Supplementary Figure S3H . Bar = 5 μm .

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(C) atg1Δpep4Δ cells containing YFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown to log phase and starved in SD‐N. Cells were monitored by time‐lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in 'Materials and methods'. Data are plotted as the mean±s.e.m. from at least 90 quantified cells. Additional images are shown in Supplementary Figure S3H. Bar=5 μm.


( D ) atg1Δatg13Δ cells expressing GFP ‐ tagged wild ‐ type Atg13 , and either wild ‐ type Atg1 or the Atg1 ‐ VE mutant ( left panel ) and wild ‐ type cells containing Atg13 ‐ GFP and either wild ‐ type Atg13 or Atg13 ‐ FV were grown to mid log phase followed by starvation for 6 h in SD ‐ N media . GFP cleavage was analysed by western blotting . Figure source data can be found with the Supplementary data .

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

(D) atg1Δatg13Δ cells expressing GFP‐tagged wild‐type Atg13, and either wild‐type Atg1 or the Atg1‐VE mutant (left panel) and wild‐type cells containing Atg13‐GFP and either wild‐type Atg13 or Atg13‐FV were grown to mid log phase followed by starvation for 6 h in SD‐N media. GFP cleavage was analysed by western blotting. Figure source data can be found with the Supplementary data.


A ) Ten - fold serial dilutions from overnight cultures of wild - type and cdc14 - 1 cells dropped and grown on solid rich media or media containing MMS at 25 , 28 , 30 or 33ºC . Note that cdc14 - 1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild - type cells .

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A) Ten-fold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33ºC. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild-type cells.


B ) Ten - fold serial dilution from mid - log phase cultures of wild - type and cdc14 - 1 cells grown on solid rich media or media containing mock DMSO ( as non - treated control ) , 4NQO , HU , phleomycin and benomyl at 30ºC . Note that cdc14 - 1 cells present a market sensitivity to all DNA damaged agents tested .

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B) Ten-fold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30ºC. Note that cdc14-1 cells present a market sensitivity to all DNA damaged agents tested.


C ) Left panel : Schematic representation showing relevant genomic structure of the strain used to assess intra - chromosomal repair . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of intra - chromosomal repair in wild - type and cdc14 - 1 cultures at the semipermisive temperature . After DSB formation by the expression of the HO , glucose was added to repress it , thus allowing repair with HM donor sequences . Genomic DNA was digested with StyI , separated on agarose gel and blotted . Blots were hybridized with a probe corresponding to the MATa - distal sequence . A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time - point . Immunoblot analysis of Rad53 during mating type switching experiments is shown . Coomassie blue staining is depicted as loading control . Graphs show quantification of gene conversion ( GC ) leading to the re - establishment of MATa or switching to MAT and DSB formation . The data was normalized with the actin loading control . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

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

C) Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermisive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time-point. Immunoblot analysis of Rad53 during mating type switching experiments is shown. Coomassie blue staining is depicted as loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MAT and DSB formation. The data was normalized with the actin loading control. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Left panel : Schematic diagram displaying relevant genomic structure of the strains used to measure inter - chromosomal DNA repair efficiency . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that crossover and non - crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 mutant strains carrying the inter - chromosomal gene conversion assay . Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression , thus producing a DSB on chromosome V . Samples were taken at different time - points , genomic DNA was extracted , digested with EcoRI and analysed by Southern blot . Blots were hybridized with a MATa - only DNA sequence and an actin probe was used as loading control . Asterisk denotes an overexposed film to visualize crossover formation . Immunoblot of Rad53 was performed as previously described . Coomassie staining is shown as a control for loading . Graphs show the mean ± S . D . of gene conversion , DSB induction and crossover vs non - crossover ratio from three independent experiments . All data was normalized using actin as loading control . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

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A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


B ) Left panel : Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 cells harbouring the repair pathway choice assay . Cells were grown overnight and HO induction was attained through addition of galactose for 1 . 5 hour at semipermisive temperature . After induction of the HO , glucose was added to repress the HO expression and samples were taken to analyse repair efficiency . DNA was extracted , digested with EcoRV , separated on agarose gels and blotted . Blots were hybridized with a probe corresponding to the MATa - only DNA sequence . A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time - point . Graphs show quantification of mating type switching by gene conversion ( HR ) , restoration of MATa ( NHEJ ) and the kinetics of the DSB induction . All data was normalized with the HIS3 gene . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown . Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment . Detection of Net1 phosphorylation levels was achieved by using Phos - tag polyacrylamide gels . Coomassie staining is showed as loading control .

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A) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown. Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment. Detection of Net1 phosphorylation levels was achieved by using Phos-tag polyacrylamide gels. Coomassie staining is showed as loading control.


A ) Live - cell imaging of Cdc14 - YFP and Cnm67 - RFP asynchronous cells treated with the DNA damage agent phleomycin . Cells were grown overnight and phleomycin was added to a final concentration of 1μM . Samples were taken at different intervals to determine Cdc14 localization . Cnm67 was used as SPB reporter to determine spindle length . Scale bar : 3μm . Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase ( left graph ) and with Cdc14 signal at the nucleoplasm ( right graph ) .

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A) Live-cell imaging of Cdc14-YFP and Cnm67-RFP asynchronous cells treated with the DNA damage agent phleomycin. Cells were grown overnight and phleomycin was added to a final concentration of 1μM. Samples were taken at different intervals to determine Cdc14 localization. Cnm67 was used as SPB reporter to determine spindle length. Scale bar: 3μm. Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase (left graph) and with Cdc14 signal at the nucleoplasm (right graph).


B ) Western blot analysis showing Net1 phosphorylation through the cell cycle is included . The separation of the phospho - bands was achieved by using Phos - tag polyacrylamide gels as in Fig 3A . Coomassie staining is depicted as loading control .

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B) Western blot analysis showing Net1 phosphorylation through the cell cycle is included. The separation of the phospho-bands was achieved by using Phos-tag polyacrylamide gels as in Fig 3A. Coomassie staining is depicted as loading control.


B ) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage . A strain containing Cdc14 - GFP and Net1 - HA was blocked in G1 by using the pheromone alpha - factor and released into the cell cycle . Samples were taken at different time - points to analyse Cdc14 spatial localization and Net1 phosphorylation levels . A representative picture of non - released ( T0 ) , partially released ( T60 ) and fully released ( T75 ) Cdc14 from the nucleolus is shown . Scale bar : 3μm . Graph represents the percentage ± SD from three independent experiments of cells with no released , partially released ( FEAR ) and totally released ( MEN ) Cdc14 from the nucleolus along the cell cycle .

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B) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage. A strain containing Cdc14-GFP and Net1-HA was blocked in G1 by using the pheromone alpha-factor and released into the cell cycle. Samples were taken at different time-points to analyse Cdc14 spatial localization and Net1 phosphorylation levels. A representative picture of non-released (T0), partially released (T60) and fully released (T75) Cdc14 from the nucleolus is shown. Scale bar: 3μm. Graph represents the percentage ± SD from three independent experiments of cells with no released, partially released (FEAR) and totally released (MEN) Cdc14 from the nucleolus along the cell cycle.


C ) Cells expressing Cdc14 - YFP and Cnm67 - RFP were grown overnight and blocked in G2 / M by using nocodazole prior phleomycin treatment ( 1μM ) . Samples were taken every 30 minutes to follow Cdc14 re - localization . The efficiency of the arrest was determined as in Fig 3A . Scale bar : 3μm . Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion ( left graph ) and cells arrested in metaphase ( right graph ) from three independent experiments .

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C) Cells expressing Cdc14-YFP and Cnm67-RFP were grown overnight and blocked in G2/M by using nocodazole prior phleomycin treatment (1μM). Samples were taken every 30 minutes to follow Cdc14 re-localization. The efficiency of the arrest was determined as in Fig 3A. Scale bar: 3μm. Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion (left graph) and cells arrested in metaphase (right graph) from three independent experiments.


D ) Live - cell imaging of Cdc14 localization in the presence of a non - reparable DSB at the MAT locus . Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media , thus producing a DSB on chromosome III . Cdc14 , the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 ( used as DSB reporter ) were labelled with the green , red and cyan fluorescent proteins respectively . Micrographs display the maximum projection of 9 planes showing Cdc14 co - localizing with the DSB and the SPBs in response to DNA damage ( arrows ) . Scale bar : 3μm . Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction ( left graph ) and the percentage of Cdc14 - SPB - DSB co - localization after 5h in galactose ( right graph ) .

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D) Live-cell imaging of Cdc14 localization in the presence of a non-reparable DSB at the MAT locus. Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media, thus producing a DSB on chromosome III. Cdc14, the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 (used as DSB reporter) were labelled with the green, red and cyan fluorescent proteins respectively. Micrographs display the maximum projection of 9 planes showing Cdc14 co-localizing with the DSB and the SPBs in response to DNA damage (arrows). Scale bar: 3μm. Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction (left graph) and the percentage of Cdc14-SPB-DSB co-localization after 5h in galactose (right graph).


E ) Western blot showing Net1 - 6HA phosphorylation state before and after the generation of a DSB induced by the HO expression . A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control . The phospho - bands were resolved by using Phos - tag polyacrylamide gels as in Fig 3A and Fig 3B . Coomassie staining is depicted as loading control .

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E) Western blot showing Net1-6HA phosphorylation state before and after the generation of a DSB induced by the HO expression. A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control. The phospho-bands were resolved by using Phos-tag polyacrylamide gels as in Fig 3A and Fig 3B. Coomassie staining is depicted as loading control.


A ) Diagram showing the genomic background and the approximation used to measure DSB - SPB interaction . Cnm67 - RFP and Ddc2 - GFP were used as SPB and DSB markers respectively . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . Graph represents the mean ± SD of D1 , D2 and D3 distances from three independent experiments . At least 100 cells per experiment were scored using the maximum projection of three z - planes . P values were calculated using a 2 - tailed unpaired Student t test . A representative picture of a wild - type strain depicting DSB - SPB interaction is shown . Scale bar : 3μm .

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A) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Cnm67-RFP and Ddc2-GFP were used as SPB and DSB markers respectively. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. Graph represents the mean ± SD of D1, D2 and D3 distances from three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture of a wild-type strain depicting DSB-SPB interaction is shown. Scale bar: 3μm.


B ) Time - lapse experiments in living cells showing the efficiency of DSB - SPB interaction upon induction of a single DSB . Cnm67 - CFP and Ddc2 - RFP were used as SPB and DSB markers respectively . D1 , D2 and D3 distances in wild - type and cdc14 - 1 cells were tracked using the maximum projection of three z - planes images at 15 - seconds intervals over period of 7 . 5 minutes . A total of 32 time - lapses for each strain were measured . A representative picture is shown . Examples of cells defective in DSB - SPB interaction are marked with arrows . Scale bar : 3μm .

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B) Time-lapse experiments in living cells showing the efficiency of DSB-SPB interaction upon induction of a single DSB. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. D1, D2 and D3 distances in wild-type and cdc14-1 cells were tracked using the maximum projection of three z-planes images at 15-seconds intervals over period of 7.5 minutes. A total of 32 time-lapses for each strain were measured. A representative picture is shown. Examples of cells defective in DSB-SPB interaction are marked with arrows. Scale bar: 3μm.


C ) An RFP - marked I - SceI recognition site is re - localized to the SPBs after expressing the endonuclease . Cells harbouring a TetO array adjacent to the I - SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC - Ade prior galactose induction . To check reproducibility with previous results a Rad52 - CFP was used . Cnm67 was labelled with the GFP to be used as SPB reporter . D1 , D2 and D3 distances were measured in wild - type and cdc14 - 1 mutants before and after endonuclease expression by using both TetI - RFP and Rad52 - CFP foci using the maximum projection of nine z - planes images . Graphs represent the average of three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . A representative picture of a wild - type strain depicting TetI - Rad52 - SPB interaction is shown . Scale bar : 3μm .

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C) An RFP-marked I-SceI recognition site is re-localized to the SPBs after expressing the endonuclease. Cells harbouring a TetO array adjacent to the I-SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC-Ade prior galactose induction. To check reproducibility with previous results a Rad52-CFP was used. Cnm67 was labelled with the GFP to be used as SPB reporter. D1, D2 and D3 distances were measured in wild-type and cdc14-1 mutants before and after endonuclease expression by using both TetI-RFP and Rad52-CFP foci using the maximum projection of nine z-planes images. Graphs represent the average of three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. A representative picture of a wild-type strain depicting TetI-Rad52-SPB interaction is shown. Scale bar: 3μm.


D ) DSB - SPB tethering requires the N - terminus domain of Mps3 . Cells expressing a wild - type Mps3 , a mps3Δ2 - 64 and a mps3Δ75 - 150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB - SPB tethering in both wild - type and cdc14 - 1 backgrounds . Ddc2 - YFP was used as DSB reporter . Measurements were carried out using a maximum projection of nine z - planes images . Graphs represent the average distribution of D1 , D2 and D3 of three independent experiments . At least 100 cells for each experiment were scored . P values were calculated using a 2 - tailed unpaired Student t test .

D) DSB-SPB tethering requires the N-terminus domain of Mps3. Cells expressing a wild-type Mps3, a mps3Δ2-64 and a mps3Δ75-150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB-SPB tethering in both wild-type and cdc14-1 backgrounds. Ddc2-YFP was used as DSB reporter. Measurements were carried out using a maximum projection of nine z-planes images. Graphs represent the average distribution of D1, D2 and D3 of three independent experiments. At least 100 cells for each experiment were scored. P values were calculated using a 2-tailed unpaired Student t test.


E ) DSB - SPB interaction involves a proficient DNA damage checkpoint activation . D1 , D2 and D3 distances were scored in wild - type , sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2 - GFP and Spc110 - RFP as DSB and SPB markers respectively . Measurements were performed by using a maximum projection of nine z - planes images . Graphs denote the average distribution of D1 , D2 and D3 from three independent experiments where at least 100 cells for each sample were scored . P values were calculated using a 2 - tailed unpaired Student t test .

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E) DSB-SPB interaction involves a proficient DNA damage checkpoint activation. D1, D2 and D3 distances were scored in wild-type, sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2-GFP and Spc110-RFP as DSB and SPB markers respectively. Measurements were performed by using a maximum projection of nine z-planes images. Graphs denote the average distribution of D1, D2 and D3 from three independent experiments where at least 100 cells for each sample were scored. P values were calculated using a 2-tailed unpaired Student t test.


A ) Identification of Cdc14 phospho - targets during the DNA damage response by mass spectrometry analysis . Wild - type and cdc14 - 1 cells were grown overnight and blocked in G2 / M by using nocodazole to avoid cell cycle - dependent changes in protein phosphorylation between both strains . After the block was attained , cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h . Differential phosphorylation of phospho - peptides detected between the wild - type and cdc14 - 1 were grouped into broad categories depending on the molecular function of the proteins . The table includes the DNA damage and checkpoint - related proteins with Cdc14 - dependent hyper - phosphorylated status and the relative ratio between the wild - type and cdc14 - 1 during the DNA damage response . Red and blue indicate relative amount of the residue phosphorylation between both strains ( Red , high ; Blue , low ) .

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A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low).


B ) Cdc14 dephosphorylates Spc110 in response to a DSB . Spc110 was tagged with the 6HA epitope in wild - type , cdc14 - 1 and spc110S36 - 91A backgrounds . Cells were grown overnight in raffinose - containing media and supplemented with galactose to induce HO expression . Samples were collected at each time - point indicated . Proteins were TCA extracted , separated on Phos - Tag containing gels and blotted . Coomassie staining is shown as loading control .

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B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control.


C ) Lambda - phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response . Cells were grown overnight in raffinose containing media ( line 1 ) and incubated with galactose for 2 . 5 hours to induce HO expression ( line 2 ) . Extracts from raffinose - containing cultures were treated with mock ( line 3 ) or λ - PPase ( line 4 ) . All samples were separated in a Phos - Tag containing gel and blotted . Coomassie staining is shown as loading control .

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C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control.


D ) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response . Spc110 was labelled with the 3HA tag in both wild - type and esp1 - 1 background strains . Cells were grown overnight in raffinose - containing media and supplemented with galactose to induce the HO expression . Samples were collected at each time - point indicated . Proteins were TCA extracted , separated on Phos - Tag containing gels and blotted . Coomassie staining is shown as a control for loading .

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D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.