A . Wild - type ( WT ) , rbd2Δ or sre1Δ yeast ( 200 cells ) containing either empty vector ( EV ) or a plasmid expressing sre1N ( Sre1amino acids 1 - 440 ) were grown on rich medium in the presence or absence of cobalt chloride ( CoCl2 ) .

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A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).


B . Western blot was probed with anti - Sre1 IgG of phosphatase - treated , whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen . Dsc5 serves as a loading control and was detected by chemiluminescence . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre1 cleavage . Western blot was performed using anti - Sre1 or anti - FlagIgG of phosphatase - treated , whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen .

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C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.


D . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre2 cleavage by western blot probed with anti - Sre2 serum or anti - FlagIgG of phosphatase - treated , whole cell lysates from cells grown in the presence of oxygen .

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D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.


E . Sre1 cleavage assay was performed as in ( C ) . G128V , A127D and A186T mutant strains were obtained from our previous mutagenesis screening ( Stewart et al , 2012 ) . Dsc5 serves as a loading control .

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E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.


F . Sre2 cleavage assay for indicated yeast strains was performed as in ( D ) .

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F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).


G . A model substrate of Sre2 containing amino acids 423 - 793 ( Sre2MS ) with an N - terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ , dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains . Whole cell lysates from indicated yeast strains were analyzed by western blot with anti - Flag antibody . Three independent isolates ( A - C ) are shown for each strain . Empty vector ( EV ) transformation serves as a negative control .

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G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.


A . Wild - type ( WT ) and the indicated mutant strains were grown for 3 hr at the non - permissive temperature ( 36 ° C ) to inactivate Mts3 . Phosphatase - treated cell lysates were analyzed by western blot with anti - Sre2 serum and imaged using chemiluminescence . P and N denote Sre2 precursor and cleaved nuclear forms , respectively .

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A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.


B . WT , rbd2Δ and sre2Δ cells were treated with bortezomib ( Bz ) for 3 hr , and phosphatase - treated , whole cell lysates were analyzed by western blot with anti - Sre2 serum .

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B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.


C . WT , dsc1Δ and rbd2Δ cells carrying either empty vector ( EV ) or a plasmid expressing 3xFlag - Sre2MS ( MS ) were treated with bortezomib ( Bz ) for 3 hr . Whole cell lysates were analyzed by western blot with anti - Flag antibody .

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C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.


D . Whole cell lysates from the indicated strains were analyzed by western blot with anti - Sre2 serum .

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D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.


E . Catalytically dead Dsc1 E3 ligase ( dsc1 - C634A ) mutant and other indicated yeast strains were analyzed by western blot probed with anti - Sre2 serum and imaged using chemiluminescence .

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E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.


A . HumanHEK293T cells were transfected with plasmids expressing different 3xHA - tagged rhomboid proteases : S . pomberbd2 , S . pomberbd2 recoded for human expression , humanRHBDL2 , D . melanogaster Rho1 , and P . falciparumROM4 . Whole cell lysates were analyzed by western blot to detect the HA - tagged rhomboid [ asterisks indicate loading control bands detected with antibody ab179726 ( abcam ) ] .

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A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].


B . HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S . pombe rbd2 , D . melanogaster Rho1 , or P . falciparumROM4 . Conditioned media and cell lysates were analyzed by western blot . P and C denote precursor and cleaved products , respectively .

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B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.


C . HumanHEK293T cells were transfected with plasmids expressing the GFP - TatA - Flag substrate and the indicated rhomboid proteases . Whole cell lysates were analyzed by western blot . P , CN , CC denote precursor , N - terminal cleaved and C - terminal cleaved products , respectively .

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C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.


D . HumanHEK293T cells were transfected with plasmids expressing the indicated GFP - TatA - Flag substrates and either wild - type or mutant recoded S . pomberbd2 . Whole cell lysates were analyzed by western blot .

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D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot.


E . HumanHEK293T cells were transfected with plasmids expressing the indicated GFP - TatA - Flag substrates and different rhomboid proteases . Whole cell lysates were analyzed by western blot .

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E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.


F . HumanHEK293T cells were transfected with plasmids expressing the GFP - TatA ( A8L ) - Flag and recoded S . pomberbd2 . C - terminal cleavage products were purified using anti - Flag antibodies and purified protein was analyzed by mass spectrometry .

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F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.


B . Whole cell lysates from WT , rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P and N denote Sre2MS precursor and cleaved forms , respectively .

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B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.


D - E . Whole cell lysates from WT , rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P and N denote Sre2MS precursor and cleaved forms , respectively . Asterisks indicate non - specific , loading control bands , which were used to normalize the intensities of P and N in Figure 5F . Two independent isolates , A and B , are shown for each strain .

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D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.


F . Intensities of individual P and N bands from western blots were quantified using LI - COR software . Relative intensity of P and N to corresponding non - specific band ( asterisk ) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis . The ratio of N to P or relative P level was presented in bar graphs .

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F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.


G . The average ratio of N to P level for different Sre2MS mutants in wild - type cells ( Appendix Fig S2 ) was plotted . Cleavage was scored as normal if the ratio was > 2 .

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G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.


H . For Sre2MS mutants showing defects in cleavage , the average fold - increase in P level in dsc1Δ cells compared to WT cells from two isolates ( Appendix Fig S2 ) was plotted . Mutants were grouped into three phenotypic classes ( intermediate , dsc1Δ , and rbd2 ∆ ) according to observed precursor accumulation . Precursor accumulation was defined as dsc1Δ if < 1 . 5 ( orange ) , rbd2Δ if > 3 ( pink ) , and intermediate if in between ( blue ) .

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H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).


B . Recombinant proteins GST - fused Rbd2 C - terminus ( Rbd2200 - 251 ) , Dsc5 UBX ( Dsc5323 - 425 ) , Dsc2 UBA ( Dsc2298 - 372 ) , and GST - HA - V5 control were bound to GST magnetic beads and incubated with S . pombe cytosol fraction from wild - type cells . Equivalent amounts of unbound and bound fractions were probed for anti - Cdc48 , anti - ubiquitin and anti - GSTIgG .

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B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG.


C . GSTpulldown assay was performed as in ( B ) using truncated forms of Rbd2 C - terminus ; Rbd2200 - 251 , Rbd2200 - 225 , Rbd2225 - 251 and Rbd2200 - 240 . Unbound ( 1x ) and bound ( 5x ) fractions were analyzed by western

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C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting


D . Single residue mutants of conserved glycine residues in Rbd2200 - 251 ( G244R and G246R ) were tested for Cdc48 binding as in ( C ) . Western blots ( B - D ) were imaged using chemiluminescence .

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D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.


A . Detergent - solubilized yeast extracts from cells containing chromosomal Flag - tagged rbd2 or rbd2 - G246R in rbd2Δ background were prepared , and proteins associated with Rbd2 - Flag were immunopurified using anti - Flag magnetic beads . Equal amount of total ( lanes 1 - 3 ) along with 25x bound fractions ( lanes 4 - 6 ) were analyzed by western blot with anti - Cdc48 serum and anti - FlagIgG .

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A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-FlagIgG.


C . Biotin - phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods . WT cells or rbd2Δ cells carrying rbd2 - Flag - APEX2 ( rbd2 - F - AX ) or rbd2 - G246R - Flag - APEX2 ( G246R - F - AX ) plasmid were lysed after biotin - labeling reaction , and proteins were denatured by heating the cells in lysis buffer containing 1 % SDS . Biotinylated proteins were then enriched using streptavidin magnetic beads . Lysates and 50x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Cdc48 serum , or anti - FlagIgG .

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C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.


A . Yeast strains ( 200 cells ) containing chromosomal Flag - tagged rbd2 , rbd2 - G244R , rbd2 - G246R or rbd2 - S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride ( CoCl2 ) .

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A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


B . Western blot of phosphatase - treated , whole cell lysates from strains in ( A ) grown for 3 hr in the presence or absence of oxygen was probed with anti - Sre1 or anti - Flag IgG . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Sre2 cleavage was assayed by western blot using anti - Sre2 serum or anti - Flag IgG of phosphatase - treated , whole cell lysates from the indicated strains . N denotes Sre2 cleaved nuclear form .

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C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.


D . Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag - Sre2MS were analyzed by western blot with anti - Flag antibody . Two independent isolates , A and B , are shown for each strain . Asterisk denotes non - specific band .

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D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band.


F . Yeast strains containing Flag - tagged rbd2 - SHP , rbd2 - SHP * , rbd2 - G246R - SHP , and rbd2 - G246R - SHP * were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in ( E ) . Indicated yeast strains were then assayed for Sre1 cleavage as in ( B ) .

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F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).


G . Indicated strains were assayed for Sre2 cleavage as in ( C ) .

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G. Indicated strains were assayed for Sre2 cleavage as in (C).


H . Wild - type , sre2Δ or strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130S in rbd2Δ background ( lane 7 - 12 ) were treated with bortezomib ( Bz ) for 2 hr , and phosphatase - treated , whole cell lysates were analyzed by western blot with anti - Sre2 serum or anti - Flag IgG .

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H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.


A . Western blot of phosphatase - treated whole cell lysates from yeast strains carrying rbd2 - Flag - APEX2 ( rbd2 - F ) , rbd2 - G246R - Flag - APEX2 ( G246R ) or rbd2 - S130A - Flag - APEX2 ( S130A ) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti - Sre1 or anti - Flag IgG . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


B . Indicated yeast strains ( 200 cells ) containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background ( lane 3 - 5 ) or yeast strains ( 200 cells ) carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background ( lane 6 - 8 ) were grown on rich medium in the absence or presence of cobalt chloride ( CoCl2 ) .

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

B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


C . Comparison of Rbd2 protein levels from overexpression system to endogenous expression . Western blot of whole cell lysates from yeast strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background and yeast strains carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background was probed with anti - FlagIgG . For quantification , the intensity of Flag band in each lane was normalized to the non - specific band ( asterisk ) .

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

C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).


D . rbd2Δ cells expressing indicated plasmids of GFP - Anp1 or GFP - Sre2MS together with rbd2 - Flag - APEX2 , rbd2 - S130A - Flag - APEX2 or rbd2 - S130A / G246R - Flag - APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin - labeling reaction . After labeling termination , cells were lysed and denatured as described in Materials and Methods . Biotinylated proteins were then enriched using streptavidin magnetic beads . Lysates ( 1x input ) and 100x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Flag IgG , anti - Cdc48 serum , and anti - GFP IgG . Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample , then normalized to the Flag intensity in enriched sample . Relative intensities of biotinylated GFP level are shown in bar graph . Error bars denote standard deviation from three biological replicates .

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

D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.


A , B Representative time - lapse images ( A ) and kymograph ( B ) of mouse zygotes expressing H2B - RFP ( magenta ) and labelled with SiR - Tubulin ( green ) . Shown is an example of marked chromosomal displacement to the cortex . C Changes in chromosome - to - cell centre distances ( d ) over time relative to anaphase - onset ( n = 50 blastomeres ) . Data information : In ( A , time is shown as h post - metaphase and scale bar = 20 µm . In ( C , data are shown as mean ± SEM .

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 O O O O O O O O O O O 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 Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.


B , C Representative time - lapse images of embryos in which DMSO ( B ) or nocodazole ( C ) was added after anaphase completion . D Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition following DMSO - ( n = 22 blastomeres ) or nocodazole - treatment ( n = 26 blastomeres ) at anaphase completion . Data information : In ( B , C , time is shown as h post - drug addition and scale bar = 20 µm . In ( D , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

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 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

B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


E - G Representative time - lapse images of embryos in which DMSO ( E ) , Nocodazole ( F ) or Cytochalasin D ( G ) was added when nuclei had travelled to the cortex . H Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition DMSO - ( n = 20 blastomeres ) , nocodazole - ( n = 12 blastomeres ) or cytochalasin D - treatment ( n = 14 blastomeres ) after nuclei reached the cortex . Data information : In E - G ) , time is shown as h post - drug addition and scale bar = 20 µm . In H ) , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


A , B Representative time - lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase ( A , n = 15 embryos ) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin ( B , n = 22 embryos ) . C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not . Each data point represents the mean maximal d value of the two blastomeres from the same embryo ( n = 45 embryos ) . D Graph of time taken to develop to the blastocyst stage in relation to maximal d values ( n = 27 embryos ) . E Graph comparing the distribution of times taken for nuclei to re - centre in embryos that developed to the blastocyst stage or not ( n = 45 embryos ) . F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re - centre ( n = 27 embryos ) . Data information : In ( A , B ) , time is shown as h post - drug washout and scale bar = 20 µm . In ( C - F ) , data are shown as mean ± SEM . In ( C , E ) , statistical significance was assessed by Welch ' s t - test , ns : not significant . In ( D , F ) , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test , ns : not significant .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.


A Representative time - lapse image of DNA and SiR - tubulin in DMSO - and BFA - treated embryos during the first mitotic division and subsequent interphase . Time is shown as h relative to the first mitotic anaphase . B Changes in nucleus - to - cell centre distances ( d ) over time in BFA - treated embryos , relative to maximal nuclear displacement to the cortex ( n = 14 blastomeres ) . Data information : In ( A , scale bar = 20 µm .

O O O O O O O B-MEASURED_VAR 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 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

A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.


D Representative time - lapse images of the second mitotic division in DMSO - and BFA - treated embryos . Dashed white lines highlight position of nuclei shortly before NEBD . Dashed yellow lines outline daughter - cells . Time is shown as h relative to the second mitotic NEBD . E Dot plot of the duration between first mitotic division anaphase - onset and second mitotic division NEBD for DMSO - ( n = 47 blastomeres ) and BFA - treated ( n = 47 blastomeres ) embryos . F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO - ( n = 44 blastomeres ) and BFA - treated ( n = 43 blastomeres ) embryos . G Plot of the spindle - to - cell centre distances against pre - NEBD nucleus - to - cell centre distance ( n = 34 blastomeres ) . Line indicates mathematically regressed fit . Data information : In D ) , scale bar = 20 µm . In E - G , data are shown as mean ± SEM . In ( E , F , statistical significance was assessed by Mann - Whitney test , * * * * P < 0 . 0001 , ns : not significant . In ( G , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test . Two - tailed p values are reported ( * * P < 0 . 01 ) .

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 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 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

D Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).


A Representative time - lapse images of 2 - cell embryos cultured with SiR - DNA to label nuclei ( white circles ) and microinjected with oil droplets ( yellow circles ) . Time is shown as h : min relative to maximal nuclear displacement . B Plot of the distances between oil droplets and nuclei to the cell - centre over time ( n = 14 oil - nucleus pairs ) . Data information : Scale bar = 20 µm in A , In ( B ) , data are shown as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.


E Top panel : an example of fluorescent bead distribution in a 2 - cell embryo . Lower panel : an example of region imaged for bead tracking at high temporal resolution . F , G Trajectories of bead movement over a 20 s - duration in oocytes ( F , n = 217 beads from 23 oocytes ) and 2 - cell embryos ( n = 277 beads from 26 embryos ) . Each coloured line represents a single bead track . Data information : Scale bar = 20 µm in E , except 2 µm in the lower panel of 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 O O O O O O O O O O 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 Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.


B Shown is a representative 2 - cell embryo immunostained for β - tubulin ( green ) and DNA ( magenta ) at anaphase completion ( n = 8 embryos ) . C Dot plot of mean β - tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus ( cortex - facing aspect , dashed white triangle in B ) or behind the nucleus ( cleavage plane - facing aspect , dashed yellow triangle in B ) ( n = 14 embryos ) . Data information : In ( B scale bar = 20 µm . In ( C , data are shown as mean ± SEM . In ( C , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O 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 Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


D Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of dynarrestin at anaphase completion ( n = 14 embryos ) . Data information : In ( D , time is shown as h post - drug addition . In D , scale bar = 20 µm .

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

D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.


E Dot plot of maximal d values between DMSO - and dynarrestin - treated embryos ( n = 28 blastomeres per group ) . Data information : In E , data are shown as mean ± SEM . In E ) , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant .

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

E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


G Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of taxol at maximal nuclear displacement . Data information : In G , scale bar = 20 µm .

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

G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.


H Changes in nucleus - to - cell centre distances ( d ) over time relative to the time of drug addition in DMSO - ( n = 19 ) and taxol - ( n = 19 ) treated 2 - cell blastomeres ( mean ± SEM ) . Data information : In H ) , data are shown as mean ± SEM . In ( H ) , statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unremarked datapoints = not significant ) .

O O O O O O O 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

H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).


I Representative images of embryos immunostained for β - tubulin and Hoechst following the completion of live imaging ( shown in E ) in DMSO - and taxol - treated embryos ( n = 9 embryos per group ) . Time is shown as h post - drug addition . Scale bar = 20 µm . Data information : In I ) , scale bar = 20 µm .

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

I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.


A . Hematoxylin and eosin ( H & E ) - stained cross section of a human liver slice . Scale bar is representative of A - F . Image is also used in Fig . S3A . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from A . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) which is representative of B , D , and F . C . H & E - stained cross section of a human lung slice . Image is also used in Fig . S3D . D . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from C . E . H & E - stained cross section of frontal cortical region of a human brain tissue . Image is also used in Fig . S3G . F . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from E . G . Quantification of relative abundance of total glycogen calculated from the sum of CL3 - CL18 in human liver , lung , and brain . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * 0 . 01 < P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison for total glycogen . H . Glycogen structure represented by normalized chain length distribution ( ratio to CL3 ) of each human liver , lung and brain from G . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . Hematoxylin and eosin ( H & E ) - stained cross section of adenocarcinoma of the prostate , lung squamous cell carcinoma ( LUSC ) , lung adenocarcinoma ( LUAD ) , and Ewing sarcoma ( ES ) of the abdomen tumor . Annotated regions are T - tumor , S - stroma , D / G - duct / glandular , and A - adipose tissue . Scale bar is representative of A and B . Images are also used in Fig . S4A , 5D and S6G . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Images are also used in Fig . S4E , 5D , and S6G . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from tumor regions shown in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . ( Left ) Hematoxylin and eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of the shoulder tumor . Annotated regions are annotated as T - tumor and M - muscle . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A - E . B . ( Left ) H & E stained cross section of an ES of the chest wall tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of the rib tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of the abdomen tumor . Annotated regions are T - tumor , S - stroma , and A - adipose tissue . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of the bladder tumor . Annotated regions are T - tumor and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) tumor , and ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , and ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) tumor , ( top , middle ) stroma , and ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , ( bottom , middle ) stroma and ( bottom , right ) adipose tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


A . ( Left ) Hematoxylin and eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at the same time as the Ewing sarcoma ( ES ) of the tibia tumor ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - tumor , and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain length . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from the ES of the tibia tumor in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

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

A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


F . In vivo tumor growth of A673 EV or GYS1 - KO injected in the flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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

F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


G . Half maximal effective concentration ( EC50 ) of the small molecular inhibitor guaiacol in A673 cells in vitro .

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

G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.


J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) and tibial ES tumor ( bottom ) . The image displays a heatmap gradient of intensity with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

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

J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


N . Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor ( T ) and stroma ( S ) tissues . Tissues were scanned digitally using the Axio Scan . Z1 side scanner . Scale bar is shown below the image . O . Quantification of immunohistochemical laforin staining between normal tissue ( n = 3 technical replicates per region ) , tibial ES tumor ( n = 7 technical replicates per region ) , and stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison .

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

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.


P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) Tumor growth of xenografts of A673 empty vector ( EV ) and laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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 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

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


A . From left to right , Surface representation of the three - dimensional structures of C3b - MCP ( CCP1 - 4 ) ( blue ) , C3b - SPICE ( CCP1 - 4 ) ( green ) , C3b - FH ( CCP1 - 4 ) ( orange ) , C3b - CR1 ( CCP15 - 17 ) ( dark red ) , C3b - DAF ( CCP2 - 4 ) ( purple ) .

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

A. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple).


B . Cartoon representation of the five regulators . The width of the cartoon is proportional to the B - factors observed in the structural data . The B - factor coloring ( from blue to red ) is in relative scale according to the resolution of the crystal structures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures.


C . Cartoon representation of the five C3b molecules in the C3b - regulator complexes . Coloring and style as in B .

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

C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B.


D . Highlight of electron density maps ( 2Fo - Fc , blue , contour level 1 . 2σ ; Fo - Fc , green and red , contour level 3σ and - 3σ , resp . ) for the regions of the C3b - regulator complexes showing flexibility . From left to right : the putative site for MCP CCP2 , SPICE CCP4 , CR1 CCP15 , and DAF CCP2 .

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

D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.


A . " Open - book " surface representation of C3b and regulator molecules , highlighting their domain organizations using different colors as shown in the neighboring schemes . Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b . The corresponding CCP domains in each regulator described in this work are shown in the bottom - left scheme . Domains CCP1 - 2 of MCP are shown in shaded grey because they are not involved in C3b binding . The table reports the extent of the regulator contact surface on C3b and the associated buried surface area .

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 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 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

A. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area.


B . Opened view of the molecular footprint of the regulator - C3b interaction ( as shown in Fig . 2A ) , highlighting the C3b domains on the regulator surface ( top ) and the regulator domains on C3b ( bottom ) .

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

B. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).


A . Superposition of MCP and FH domains CCP3 - 4 ( shown as blue and orange cartoons , resp . ) on the surface of C3b .

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

A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b.


B . Cartoon representation of the superposition of domains CCP3 - 4 of MCP ( blue ) , FH ( orange ) and SPICE ( green ) . Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4 , MCP and SPICE show a rotation of approximately 100 ° in the position of CCP4 compared to FH .

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

B. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH.


C . The reported mutagenesis data on MCP CCP3 and CCP4 ( data from Liszewki et al . ( Liszewski et al , 2000 ) ) are consistent with the crystal structure . Shown are two orientations of the surface of MCP CCP3 - 4 as in the crystal structure of the C3b - MCP complex , highlighting the contact sites with the C3b platform ( purple ) , the MCP mutations identified as critical for C3b binding ( red ) and those not affecting the interaction ( blue ) . The GB24 antibody epitope is shown as yellow contour on the MCP surface .

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

C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface.


D . Model of the putative positioning of the flexible CCP1 - 2 domains of MCP . The model has been generated by superposing the structure of CCP1 - 4 of MCP from PDB 3O8E ( Persson et al , 2010 ) onto the structure of the C3b - MCP ( CCP1 - 4 ) complex . CCP1 - 2 domains of MCP , not visible in the C3b - MCP ( CCP1 - 4 ) structure , are shown in grey .

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D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey.


E . Highlight of the primary sequence differences between SPICE and VCP . The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple . The contact surface of C3b on the regulator in the C3b - SPICE ( CCP1 - 4 ) complex is shown in green on the right panel .

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

E. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.


( A ) Mapping of known disease - related mutations on C3b ( Table 3 ) . The high - resolution C3b three - dimensional structure is used as structural template , shown as white surface . The regulator contact surfaces ( based on C3b - FH ( CCP1 - 4 ) ( Wu et al , 2009 ) and C3d - FH ( CCP19 - 20 ) ( Kajander et al , 2011 ; Morgan et al , 2011 ) ) are shown as black contour . All mutations reported in Table 3 are shown in blue on the C3b surface . Disease - related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel .

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

(A) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel.


( B ) Mapping of disease - related mutations on the FH ( CCP1 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

O O O O O O O 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) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A.


( C ) Mapping of disease - related mutations on the MCP ( CCP3 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

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

(C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.


B Negative stain image of 2D crystals containing the FERM - kinase region of FAK bound to a PI ( 4 , 5 ) P2 monolayer . Top right insert : Fourier transform of the 2D crystal image . Bottom right insert : 2 . 5x zoom of the main image .

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

B Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image.


A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles ( PC ; phosphatidylcholine ) or PI ( 4 , 5 ) P2 containing vesicles . Plotted are mean values from two independent autophosphorylation reactions , each determined in duplicates ( n = 4 ) . Mutant nomenclature is as shown in Table 2 .

O O 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 I-CONTROLLED_VAR I-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 Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5)P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2.


B Catalytic turnover activity as measured in a coupled kinase assay is shown . Plotted are mean values from at least three 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

B Catalytic turnover activity as measured in a coupled kinase assay is shown. Plotted are mean values from at least three independent experiments.


C To determine whether autophosphorylation occurs in cis or trans , autophosphorylation was measured for WT or a kinase dead form of FAK ( KD , contains K454R mutation ) in presence or absence of a substrate deficient FAK mutant ( Y397F ) and / or PI ( 4 , 5 ) P2 vesicles . PI ( 4 , 5 ) P2 negative experiments contain control PC vesicles instead . Note that the scales of the two plots are different , i . e . autophosphorylation is increased in presence of PI ( 4 , 5 ) P2 . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) . Left : Schematic of possible cis - and trans - autophosphorylation ( red arrows ) in mixtures of FAK - WT / FAK - Y397F and FAK - KD / FAK - Y397F .

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

C To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5)P2 vesicles. PI(4,5)P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5)P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F.


D Src phosphorylation of Y577 in FAK in presence of PC or PI ( 4 , 5 ) P2 vesicles as determined by an ELISA method . The plot on the left is obtained in presence Src containing only the kinase domain ( Src254 - 536 ) and the plot on the right with Src containing additionally the SH3 and SH2 domains ( Src84 - 536 ) . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) .

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

D Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5)P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4).


Interactions between WT or interface mutant FAK with a PI ( 4 , 5 ) P2 containing membrane was probed using a pulldown assay with membrane coated silica beads . Plotted are bound protein relative to the binding for WT at the highest PI ( 4 , 5 ) P2 concentration . Mutant naming is as in Table 2 . For WT the non - specific binding to a PC membrane is shown for comparison . Plotted are mean values from at least three independent experiments . Error bars : s . d .

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

Interactions between WT or interface mutant FAK with a PI(4,5)P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5)P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d.


A FAK phosphorylation of WT or interface mutant FAK in SCC cells . Whole cell lysates were subjected to western blot analysis with anti - pFAK Y397 , anti - pFAK Y576 / 577 , anti - pFAK Y861 , anti - pFAK Y925 and anti - FAK . Anti - GAPDH served as a loading control . The graph shows densitometric analysis of relative pFAK / FAK ratios ( mean ) from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

O B-MEASURED_VAR 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 B-MEASURED_VAR 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 O

A FAK phosphorylation of WT or interface mutant FAK in SCC cells. Whole cell lysates were subjected to western blot analysis with anti-pFAK Y397, anti-pFAK Y576/577, anti-pFAK Y861, anti-pFAK Y925 and anti-FAK. Anti-GAPDH served as a loading control. The graph shows densitometric analysis of relative pFAK/FAK ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


B Downstream signaling in SCC cells expressing WT or interface mutant FAK . Whole cell lysates are analyzed by western blot analysis with the indicated antibodies . The graph shows densitometric analysis of relative phospho / total protein ratios ( mean ) from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

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

B Downstream signaling in SCC cells expressing WT or interface mutant FAK. Whole cell lysates are analyzed by western blot analysis with the indicated antibodies. The graph shows densitometric analysis of relative phospho/total protein ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


C Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK , Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti - pSrc Y416 , anti - FAK , anti - Src or non - specific IgG - agarose , followed by western blot analysis with anti - FAK , anti - pSrc Y416 and anti - Src . Anti - GAPDH served as a loading control . For full blots see source data .

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

C Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK, Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti-pSrc Y416, anti-FAK, anti-Src or non-specific IgG-agarose, followed by western blot analysis with anti-FAK, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. For full blots see source data.


FAK - expressing SCC cells were grown on glass coverslips , fixed and stained with anti - FAK anti - Paxillin and DAPI Representative immunofluorescence images are shown . Scale bars , 20 μm .

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FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm.


B FAK - expressing SCC cells were grown on glass coverslips , fixed and stained with anti - pSrc Y416 , anti - Paxillin and DAPI Representative immunofluorescence images are shown . Scale bars , 20 μm . The graph ( B ) shows the quantification of internalized active Src from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars , s . d . * = p < 0 . 01 .

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 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 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

B FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416 , anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01.


C Subcellular fractionation of SCC cells expressing FAK - WT or interface mutant FAK . Whole cell lysates ( WCL ) and subcellular fractions were subjected to western blot analysis with anti - FAK , anti - Tubulin ( marker for cytoplasmic fraction ; C ) , anti - RCAS ( marker for perinuclear fraction ; PN ) and anti - H4 ( marker for nuclear fraction ; N ) . The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

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

C Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


A FAK - WT , interface mutant FAK or FAK - / - SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation . Images were taken from 10 random fields after nine days ( representative images are shown ) and the relative colony size ( top graph ) as well as the number of colonies ( bottom graph ) were assessed from three independent experiments . Scale bars , 200 μm . Student ' s t - test was carried out to calculate the statistical significance . Error bars s . d . * = p < 0 . 01 , # = p < 0 . 05 .

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

A FAK-WT, interface mutant FAK or FAK -/- SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation. Images were taken from 10 random fields after nine days (representative images are shown) and the relative colony size (top graph) as well as the number of colonies (bottom graph) were assessed from three independent experiments. Scale bars, 200 μm. Student's t-test was carried out to calculate the statistical significance. Error bars s.d. * = p < 0.01, # = p < 0.05.


B FAK - WT , interface mutant FAK or FAK - / - SCC cells were seeded on growth factor - reduced Matrigel in serum - free media . After 72 h , invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel . Representative images of the invasion assay are shown . Scale bars , 200 μm . The graph shows relative invasion from five independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars , s . e . m . * = p < 0 . 01 , # = p < 0 . 05 .

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

B FAK-WT, interface mutant FAK or FAK -/- SCC cells were seeded on growth factor-reduced Matrigel in serum-free media. After 72 h, invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel. Representative images of the invasion assay are shown. Scale bars, 200 μm. The graph shows relative invasion from five independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.e.m. * = p < 0.01, # = p < 0.05.


( A ) Representative immunofluorescence images of Polo - phospho ( ph ) - T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression . Insets show magnifications of the outlined regions showing kinetochores ( KTs ) in early prometaphase ( EP ) , late prometaphase ( LP ) KTs and aligned KTs in metaphase ( M ) . Plotted profiles of signal intensities of phospho ( ph ) - T182 and the KT protein Spc105 are shown for the highlighted KTs . ( B ) Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in ( A ) . phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP , which was set to 100 % ( n ≥ 67 KTs from at least 19 neuroblasts for each condition , n = 4 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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 O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images of Polo-phospho(ph)-T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression. Insets show magnifications of the outlined regions showing kinetochores (KTs) in early prometaphase (EP), late prometaphase (LP) KTs and aligned KTs in metaphase (M). Plotted profiles of signal intensities of phospho(ph)-T182 and the KT protein Spc105 are shown for the highlighted KTs. (B) Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in (A). phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP, which was set to 100% (n≥67 KTs from at least 19 neuroblasts for each condition, n=4 independent experiments).Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.