( A ) Top , content ( n / 2n ) of exponentially growing ( OD600 = 0 . 6 - 0 . 8 ) smc2 - 8 yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . Graph : Pkn of YEp13 before after inactivation of condensin .

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(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.


Top , content ( n / 2n ) of exponentially growing yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . ( B ) Experiments conducted as in ( A ) but in scc1 - 73 yeast . Graph : Pkn of YEp13 before after inactivation of cohesin .

Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.


Top , content ( n / 2n ) of exponentially growing yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . ( C ) Experiments conducted as in ( A ) but in smc6 - 9 yeast . Graph : Pkn of YEp13 before after inactivation of Smc5 / 6 .

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Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.


( D , E ) Experiments conducted as in ( A ) , but in arrested in G1 with alfa - factor ( D ) or in metaphase with nocodazole ( E ) for 2 h at 26oC for one additional hour at 26oC or 35oC .

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(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


( F , G ) Experiments conducted as in ( B ) , but in arrested in G1 with alfa - factor for 2 h at 26oC ( F ) or in metaphase with nocodazole ( G ) for one additional hour at 26oC or 35oC .

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(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.


( H , I ) Experiments conducted as in ( C ) , but in arrested in G1 with alfa - factor ( H ) or in metaphase with nocodazole ( I ) for 2 h at 26oC for one additional hour at 26oC or 35oC .

(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


( A - E ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of condensin ( 35oC ) in smc2 - 8 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of condensin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t ) : ns , p > 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001.


( F ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of condensin .

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(F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.


( A - D ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of cohesin ( 35oC ) in scc1 - 73 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of cohesin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t ) : ns , p > 0 . 05 .

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(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.


( E ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of cohesin . P - values ( Student ' s t ) : * * p < 0 . 01 .

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(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.


( A - D ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of Smc5 / 6 ( 35oC ) in smc6 - 9 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of Smc5 / 6 ( mean ± SD from two independent experiments ) .

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(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).


( E ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of Smc5 / 6 .

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(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.


( a , b ) Multiple MBds associate with a PC3 ( a ) a B - lymphoblast ( b ) . Insets : MBd labelling ( a ) merged differential interference contrast microscopy image with MBd labelling to show boundaries ( b ) . MKLP1 , MBd marker ( a , b ; red ) ; CD44 , ( a ; green ) ; DAPI , ( a ; blue ) . Scale bar , 5 μm ( a ) 2 μm ( b ) .

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(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b).


( c , d ) Three - dimensional reconstructions of polarized in a monolayer ( c ) a HeLa ( d ) show intracellular MBds . ( c ) ZO - 1 , tight junction ; MKLP1 , MBds . Scale bar , 2 μm . Enlargement ( bottom panel ) of box in top panel shows five MBds ( arrows ) . ( d ) Wheat - germ agglutinin , plasma ( red ) ; MKLP1 - GFP , MBds ( green ) ; DAPI , ( blue ) . Scale bar , 5 μm .

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(c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm.


( e ) Electron microscopy of an MBd in a permeabilized MCF - 7 showing immungold labelling with MKLP1 antibodies . Inset , lower magnification of MBd ( boxed ) in ; nucleus , right . Scale bar , 200 nm .

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(e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm.


( f ) Time - lapse during extracellular trypsin treatment of HeLa show retention of most MBds ( MKLP1 - GFP , red ) . Two MBds ( yellow arrows ) are lost on treatment , indicating digestion / or dissociation . Time ( h : min ) post trypsin . Scale bar , 5 μm .

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(f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1-GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm.


( g ) Two - day co - cultures of HeLa expressing either MKLP1 - GFP ( MBd marker ) or RFP . Green MBds ( arrows ) associated with red ( asterisk ) indicate post - mitotic transfer of MBds between . Scale bar , 10 μm .

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(g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.


( a ) CETN1 - GFP signal is brighter in upper centrosome / spindle pole of a mitotic spindle . merged differential interference contrast microscopy image with CETN1 - GFP labelling at two centrosomes shows a metaphase chromosome . Insets : enlargements semi - quantitative integrated profiles of centrioles . Scale bar , 5 μm . ( b ) brighter CETN1 - GFP signal represents older centrosome , as it co - stains more intensely for hCenexin1 remains more intense throughout division ( Supplementary Fig . S1a ) . Scale bar , 5 μm . Lower left , merge .

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(a) The CETN1-GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1-GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1-GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge.


( c , d ) Time - lapse show that mitotic midbody is preferentially inherited by daughter with older centrosome in HeLa ( c ) hESCs ( d ) . were imaged at indicated times ( h : min ) from telophase by phase - contrast microscopy ( c ) from metaphase by differential interference contrast microscopy ( d ) . Middle panel of ( c ) upper left panel of ( d ) : CETN1 - GFP at centrosomes ; enlargements integrated profiles show that daughter with older centrosome ( c , upper ; d , lower ) inherits MBd ( lower right in c , d ) . Mitotic midbody MBds ( c , d ; arrows ) . MKLP1 , MBd marker ( red ) ; α - tubulin , mitotic midbody - boundary marker ( green ) ; DAPI , ( blue ) . Scale bars , 10 μm ( c , d ) .

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(c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).


( a ) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1 + in of basal layer where reside . Scale bar , 20 μm . Inset : Enlargement of marked by arrow .

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(a) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1+ puncta in cells of the basal layer where stem cells reside. Scale bar, 20 μm. Inset: Enlargement of the cell marked by the arrow.


( b , c ) Electron of mitotic midbody ( b , arrow ) multiple midbody - like structures in interphase with similar shapes sizes in a juxtanuclear position ( c , arrows ) in basal of mouse seminiferous tubules . N , nucleus . Scale bars , 1 μm .

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(b,c) Electron micrographs of mitotic midbody (b, arrow) and multiple midbody-like structures in interphase cells with similar shapes and sizes in a juxtanuclear position (c, arrows) in basal cells of mouse seminiferous tubules. N, nucleus. Scale bars, 1 μm.


( d ) Representative planes of a neural progenitor in ventricular zone ( Sox2 + , bottom left panel ) of an E13 . 5 mouse brain show that an intracellular MBd ( asterisk ) is associated with ventricle - facing daughter in asymmetrically dividing ( top row ) . bottom row emphasizes position of paired chromosomes in a dividing anaphase . CD133 , midbody / MBd marker ( green ) ; Na / K - ATPase , - border marker ( red ) ; DRAQ5 , ( blue ) ; DAPI , . v , ventricle . Scale bar , 5 μm . Note that abscission occurs apically in these .

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(d) Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Note that abscission occurs apically in these cells.


( e ) A histological section through a hair follicle ( left , phase - contrast microscopy ) stained for - marker keratin 15 ( K15 ) to identify bulge region ( dotted box ) , - niche . stain ( DAPI ) phase - contrast microscopy image show full follicle architecture . ( f ) Upper panels show MBd - accumulating in bulge region ( boxed ) co - labelled with K15 MKLP1 . Enlargements ( lower panels ) of boxed region highlight a with four MBds ( asterisks ) . N , nucleus . Scale bar , 5 μm .

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(e) A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. (f) Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm.


( g - i ) Quantitative representative show a decrease in MBd - accumulating on differentiation of pluripotent ( g ) to fibroblast - like ( h ) , an increase in MBd - accumulating after reprogramming differentiated ( h ) to induced pluripotent ( i ) . Numbers refer to mean ± s . d . , n = 3 . MKLP1 , MBds ; ZO - 1 , tight junctions ; α - tubulin , microtubules ; Aurora B , midbodies . Scale bar , 10 μm .

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(g-i) Quantitative analysis and representative images show a decrease in MBd-accumulating cells on the differentiation of pluripotent stem cells (g) to fibroblast-like cells (h), and an increase in MBd-accumulating cells after reprogramming differentiated cells (h) to induced pluripotent stem cells (i). Numbers refer to mean±s.d., n=3. MKLP1, MBds; ZO-1, tight junctions; α-tubulin, microtubules; Aurora B, midbodies. Scale bar, 10 μm.


( b ) - chased with EdU show a decrease in EdU ( x axis ) over time ( y axis ) , reflecting dilution of dye after divisions . ( c , d ) After a 96 h chase period , EdU were compared between with MBd numbers of > 1 , 1 0 ( y axis ) in HeLa ( c ) SAOS - 2 ( d ) . In both cases , no significant differences were noted ( c , P = 0 . 2101 ; d , P = 0 . 5609 , one - way of variance , with at least 800 analysed for each experiment , n = 3 ) , indicating similar cycling rates among different subpopulations of . ( b - d ) Each graph is a representative experiment . analysed are shown by green points , medians are depicted by vertical red lines , horizontal red lines with ticks illustrate interquartile range .

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(b) Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (c,d) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, P=0.2101; d, P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.


( a ) Depiction of FPP . Digitonin selectively permeabilizes plasma but not internal membranes . Proteinase K degrades components but membranous compartments remain intact . Under these conditions , MKLP1 - GFP - labelled MBds ( blue ellipse ) in cytoplasm will be degraded whereas those inside - bound compartments will not . ( b ) MBds in MBd - poor hRPE - 1 are largely protected ( ∼ 90 % in membranous compartments , 10 analysed ) , whereas most MBds in HeLa are not ( ∼ 27 % , 11 analysed ) , are thus degraded in cytoplasm . Scale bar , 5 μm .

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(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm.


( c ) Presence of MBds in lysosomes on chloroquine or E64d - pepstatin A ( E64d - PepA ) inhibition in hRPE - 1 HeLa , but not in MCF - 7 H9 hESCs . Chloroquine treatment of H9 hESCs is not included as it caused differentiation . A representative image of hRPE - 1 inhibited by chloroquine is shown , depicting two MBds inside lysosomes . MKLP1 LAMP2 are used as MBd ( red ) lysosome ( green ) markers , respectively . DAPI , ( blue ) . n = 100 MBds per treatment in each of biological triplicates . Scale bar , 5 μm .

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(c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm.


( d ) of MBd + ( MBd ) , of MBds within lysosomes of exiting cytokinesis following synchronization . MKLP1 LAMP2 are used as markers as in c . Note that MBds are transferred into only one of two nascent daughter after abscission ( Fig . 2d ) , so a 50 % maximum will be expected for MBd + . peak of MBds transferred to is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h .

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(d) Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in c. Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h.


( e ) Both chloroquine E64d - PepA treatments increase of MBd + in hRPE - 1 HeLa ( chloroquine , P = 0 . 0021 P = 0 . 0187 , respectively ; E64d - PepA , P = 0 . 0022 P = 0 . 0043 , respectively ; n = 3 for experiments ) . In contrast , lysosomal inhibition has no detectable effect on hESCs ( H1 , H9 ) or MCF - 7 cancer . DMSO , dimethylsulphoxide . Data are presented as mean ± s . d . ( c - e ) , except mean ± s . e . m . in hESCs ( e ) .

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(e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).


( a ) Single - plane confocal microscopy of MBds within LC3 - positive autophagosomes in MEFs expressing GFP - LC3 ( left ) in hRPE - 1 stained for endogenous LC3 ( right ) . MBd markers : Cep55 , MKLP1 or MgcRacGAP . Autophagosomes : GFP - LC3 or LC3 . Note that MKLP1 ( blue ) MgcRacGAP ( red ) are co - localized ( magenta ) in autophagosome ( green ) , indicating that MBds are sorted into autophagosomes . Scale bars , 2 μm .

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(a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm.


( b ) Decreasing autophagy by deletion of Atg5 gene ( left ) or depletion of ATG7 by siRNA ( right ) significantly increases of MBd + ( P = 0 . 0019 P = 0 . 021 , respectively , n = 3 ) . Immunoblots confirm loss of Atg5 - Atg12 conjugation in mutant depletion of ATG7 ( asterisk ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


( c ) Rapamycin ( Rapa ) LiCl co - treatment induces autophagy decreases of MBd + ( left , HeLa ; P = 0 . 0056 , n = 3 ) . Immunoblots showing increased LC3 - II confirm autophagy induction . Induction of autophagy by overexpression of Flag - tagged BECN1 reduces of MBd + ( right , MCF - 7 ; P = 0 . 0008 , n = 4 ) . Ctrl , control .

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(c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control.


( d ) Representative immunoblots showing high autophagy in normal low in cancer . Autophagic flux ( autophagic activity ) was measured by changes in of LC3 - II , in presence or absence of lysosomal inhibitors E64d - PepA . U , uninhibited . I , inhibited . Below , average of change in LC3 - II after lysosomal inhibition from three experiments . α - tubulin , loading control .

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(d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control.


( a ) Single - plane confocal microscopy showing co - localization of MBds autophagic receptor NBR1 in U2OS p62 - deleted MEFs . MBd markers : MKLP1 or Cep55 . Scale bar , 2 μm .

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(a) Single-plane confocal microscopy images showing co-localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Scale bar, 2 μm.


( b ) of MBd + is significantly increased following depletion of NBR1 ( P = 0 . 022 , n = 3 ) , but not another autophagic receptor , p62 . Co - depletion of NBR1 p62 does not further increase MBd over NBR1 depletion alone .

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(b) The percentage of MBd+ cells is significantly increased following the depletion of NBR1 (P=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone.


( c ) Deletion of p62 gene does not affect of MBd + . For b c , immunoblots verify loss .

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(c) Deletion of the p62 gene does not affect the percentage of MBd+ cells. For b and c, immunoblots verify protein loss.


( d ) Co - immunoprecipitation ( IP ) reveals that CEP55 NBR1 form a . Precipitated 5 % of input material ( Input ) were analysed by immunoblotting ( IB ) with antibodies against NBR1 or CEP55 .

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(d) Co-immunoprecipitation (IP) reveals that CEP55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analysed by immunoblotting (IB) with antibodies against NBR1 or CEP55.


( e - g ) Overexpression of CEP55 - eGFP ( enhanced green ) increases of MBd + ( e ; P = 0 . 0007 , n = 3 ) of NBR1 - negative MBds ( f ; P = 0 . 0568 , n = 3 ) , presumably by sequestering NBR1 ( red ) away from MBds in expressing CEP55 - eGFP ( green ) as shown in g , consequently preventing MBd degradation . dotted box in g is enlarged ( top right panel ) , labelling of NBR1 CEP55 - eGFP ( middle bottom right panels ) are also presented . DAPI , ( blue ) . Scale bar , 5 μm . data are presented as mean ± s . d . ( b , c , e , f ) . Uncropped of blots are shown in Supplementary Fig . S6 .

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(e-g) Overexpression of CEP55-eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (e; P=0.0007, n=3) and the percentage of NBR1-negative MBds (f; P=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-eGFP (green) as shown in g, and consequently preventing MBd degradation. The dotted box in g is enlarged (top right panel), and the labelling of NBR1 and CEP55-eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (b,c,e,f). Uncropped images of blots are shown in Supplementary Fig. S6.


( a - c ) Reprogramming is more efficient after MBd enrichment . Differentiated ( dH1f ) embryonic fibroblasts ( IMR90 ) are reprogrammed after stable expression of either NBR1 shRNA or shNT . Emerging iPSC colonies are scored on basis of TRA - 1 - 60 expression . depleted of NBR1 to increase MBd show an increase in iPSC colony formation ( a , b , dH1f , 3 . 1 ± 0 . 5 - fold , n = 15 , P = 0 . 00035 ; IMR90 , 3 . 4 ± 0 . 8 - fold , n = 3 , P = 0 . 02 ; data are mean ± s . e . m . ) but insignificant changes in autophagic activity ( c ) over shNT control . ( b ) Representative plates with TRA - 1 - 60 - immunostained iPSC colonies . immunoblot ( c , top ) densitometry ( c , bottom ; of autophagic flux ) show representative results ( n = 3 ) ; α - tubulin , loading control .

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(a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a,b, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (c, top) and densitometry (c, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control.


( d ) MCF - 7 side - population ( SP ) have a significantly higher of MBd + over non - side - population ( MP ; P = 0 . 0015 , n = 3 ; data are mean ± s . d . ) .

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(d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.).


( e , f ) MBd enrichment in cancer leads to increased anchorage - independent growth . MKLP1 - GFP - expressing HeLa are separated into ' MBd - high ' ' MBd - low ' subpopulations . An increase in ' MBd high ' over ' MBd low ' is associated with an increase in soft - agar colony formation ( e ) . No significant difference was observed when enrichment of MBd - high subpopulation was less than threefold . More soft - agar colonies are formed when MBds are enriched by NBR1 depletion ( NBR1 shRNA ) in HeLa ( f , left ; P = 0 . 0012 , n = 3 ) mouse 134 - 4 ( f , right ; P = 0 . 0086 , n = 3 ) ; control , shNT . Data are mean ± s . d . , colony number ( e , f ) is sum of INT - violet - stained colonies from ten random fields .

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(e,f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (f, left; P=0.0012, n=3) and mouse 134-4 cells (f, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (e,f) is the sum of INT-violet-stained colonies from ten random fields.


C - F . Polysome profiling of yeast with ( C - E ) or without ( F ) cycloheximide ( CHX ) treatment , immunoblots for Stm1 Rpl3 in polysome fractions , of Stm1 distribution across polysome profiles of grown under nutrient - rich condition ( C , F ) , rapamycin treatment for 1 hour ( D ) , or nitrogen starvation for 1 hour ( E ) . polysome profiles were fractionated on a 5 - 50 % sucrose density gradient containing 150 mM KCl into 9 fractions , precipitated immunoblotted for Stm1 Rpl3 . Rpl3 is used as a loading control . Each experiment was repeated at least three times .

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C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.


G . Polysome profiles of stm1 ∆ containing either full - or N - terminally deleted or C - terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour .

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G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.


H . Binding of full - or deletion mutants of Stm1 across polysome profile fractions shown in Figure 2E . 9 equal volume fractions of each polysome profile were precipitated analyzed by immunoblot for Stm1 Rpl3 . At least two biological replicates are used for each mutant .

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H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.


A - B . In vitro kinase ( A ) its quantitation ( B ) using recombinant mTORC1 recombinant Stm1 or Stm1AAA as a substrate in presence of 32P - γ - ATP . Immunoblots for Stm1 Raptor are shown . Asterisk indicates auto - phosphorylation of a mTORC1 component . INK128 is used as an inhibitor of mTOR . Minimum of three replicates are used for statistical by unpaired t - . Data are presented as mean ± SD .

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A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.


C . Polysome profiles of wild - type stm1 ∆ under nitrogen starvation for 1 hour . D . Polysome profiles of wild - type STM1AAA under nitrogen starvation for 1 hour . E . Polysome profiles of wild - type STM1EEE under nitrogen starvation for 1 hour . F -

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C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-


A - B . Ribosomal subunit profiles ( A ) quantitation of ribosome content ( B ) of stm1 ∆ , wild - type , STM1AAA , STM1EEE treated with rapamycin for 24 hours . For ribosomal subunit profile analyses , extracts were treated with 50 mM EDTA separated on 5 - 25 % sucrose density gradients . At least five biological replicates are used for calculation of p - values from multiple unpaired t - are indicated in graphs .

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A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.


E - G . Immunoblot of Rpl24 - GFP free GFP ( E ) , of total GFP relative to Pgk1 ( F ) , free GFP relative to Rpl24 - GFP ( G ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE treated with rapamycin for 24 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - . ( s . e . for short exposure ; l . e . for long exposure ) .

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E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).


J - K . Immunoblot ( J ) ( K ) of Rpl3 in wild - type stm1 ∆ without rapamycin , or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - .

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J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.


A - B . Puromycin incorporation ( A ) its ( B ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE starved of nitrogen for 24 hours restimulated with ( + N ) for 10 , 20 , or 30 minutes . Immunoblots for puromycin , Stm1 , Rpl3 , Pgk1 are shown . Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t - .

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A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.


D - E . Representative microscopic for expression of Rpl24 - GFP ( D ) its ( E ) in tetrads of control ( RPL24GFP ) stm1 ∆ RPL24GFP ( n = 100 , each ) . Experiments are performed at least three times . Statistical is done by unpaired t - .

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D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.


A - C . Polysome profile immunoblotting of SERBP1 , RPS6 , RPL7a across polysome fractions of HEK293T treated with DMSO ( A ) , INK128 ( B ) , or harringtonine ( C ) for 1 hour . Polysome profiles were performed using 5 - 50 % sucrose density gradients containing 150 mM KCl .

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A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.


I - J . Immunoblot for ubiquitin ( I ) its ( J ) in supernatant ribosomal pellet obtained from HEK293T treated with control sgRNA ( sgCtrl ) or SERBP1 sgRNA ( sgSERBP1 ) along with INK128 treatment for 48 hours . At least four biological replicates are analyzed by unpaired t - . Data are presented as mean ± SD .

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I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.


C , Comparison of wild - type colonies with phytochrome hox - deletion strains in presence of 0 . 12 mM menadion ( MD ) , 7 mM H2O2 or 2 mM tert - butyl hydroperoxide ( tBooH ) . strains were incubated for 5 days at 28 ° C in .

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C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.


E , Overlay of normalized ( at 705 nm ) difference spectra plus a difference spectrum of photosensory domain in presence of HO

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E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO


B , HoxA - GFP CitA - GFP ( ca . 170 from N - terminus of citrate synthase ) were expressed over night at 30 ° C in minimal medium with 2 % threonine 0 . 2 % glucose . Mitochondria were incubated with 100 μg / ml proteinase K ( PK ) for 20 min on ice . Crude extract ( CE ) , supernatant 1 ( S1 ) containing mitochondria , pellet 1 ( P1 ) , supernatant 2 ( S2 ) pellet 2 ( P2 ) with mitochondria were analyzed by Western Blot using anti GFP antibodies . CitA - GFPc = form ; CitA - GFPi = imported version .

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B, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.


C , D Role of C - terminal anchor in HoxA . Microscopy was done as in A using a microscope without Airyscan technology . C , Expression of GFP - HoxA lacking C - terminal anchor ( ∆ CTA ) in A . alternata . D , Expression of GFP fused to CTA motif .

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C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.


A , BiFC ( split - YFP ) of HOs phytochrome . Strains ( SChS29 - 33 ) were grown over night at 28 ° C minimal medium with 2 % threonine 0 . 2 % glucose . MitoTracker Red CMOXRos was used for mitochondrial . mitotracker signal was observed in RFP split - YFP signal in YFP channel of microscope . Pictures in both channels were overlaid ( merge ) . left pictures show same hyphae in differential interference contrast ( DIC ) . scale bar represents 10 µm .

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A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.


C , Co - expression of HOXs along with photosensory domain of A . nidulans FphA ( PGP ) over night at 20 ° C . PGP was purified via Strep - tag system eluate concentrated ( upper panel ) . lanes were loaded as follows : negative control before induction ( - ) , 20 h after induction ( 20 h ) crude extract ( CE ) , supernatant ( S ) , pellet ( P ) , flow through ( F ) eluate ( E ) . fractions were analyzed by Western Blot using Anti - His antibody ( lower panel ) .

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C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).


A , B , Biolayer interferometry with Apo - PGP A holo - PGP B . were immobilized at a streptavidin sensor tip . Free binding sites were blocked with biotin . Association dissociated kinetics were recorded at indicated HoxA concentrations . Kd values were calculated with manufacturers software using a global fit for both

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A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment


( B ) BW25113 ( ycbB , relA ' ) its derivatives obtained by deletion of endopeptidase genes . BW25113 ( ycbB , relA ' ) is an abbreviated name for BW25113 ΔrelA pKT2 ( ycbB ) pKT8 ( relA ' ) .

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(B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , its derivatives harboring plasmids encoding MepM , MepM H393A , MepM ΔLytM were tested in presence of ceftriaxone at 8 µg / ml ( + ceftriaxone ) or in absence of drug ( - ceftriaxone ) in BHI agar plates supplemented with 40 µM IPTG 1 % L - arabinose for induction of ycbB relA ' , respectively . genes encoding MepM , MepM H393A , MepM ΔLytM were inserted into vector pHV9 under control of PphlF promoter , which is inducible by 2 , 4 - diacetylphloroglucinol ( DAPG ) . Basal level of expression of mepM under control of PphlF was sufficient to restore ceftriaxone resistance in absence of inducer .

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Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , its derivatives harboring plasmids encoding MepM ,

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Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM


mepM gene was expressed under control of TIS1 or TIS2 ( translation initiation ) of PrhaBAD ( L - rhamnose - inducible promoter of vector pHV30 ) . Growth of BW25113 ( ycbB , relA ' ) ΔmepM harboring pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg / ml , IPTG 40 µM ( induction of ycbB ) L - arabinose 1 % ( induction of relA ' ) . ( A ) For TIS2 , growth around paper disks containing 1 or 2 mg of L - rhamnose indicated that induction of expression of mepM was required for ceftriaxone resistance . In contrast , a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in absence of inducer . ( B ) experiment was repeated with pre - exposed to L - rhamnose that were harvested at vicinity of disk containing 2 mg of L - rhamnose . Expression of ceftriaxone resistance remained dependent on presence of L - rhamnose indicating that requirement for MepM is not transient . diameter of growth zones is larger in panel ( B ) than in panel ( A ) as expected from fact that in inoculum used in ( B ) had been grown in presence of inducer already contained MepM . In ( A ) growth is only possible after diffusion of L - rhamnose in medium prior to action of ceftriaxone . At a from disk , diffusion was not sufficiently rapid to observe resistance .

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The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.


( A ) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase digested by lysozyme ( upper panel ) or by lysozyme 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

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(A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).


Functional complementation of mepM deletion in BW25113 ( ycbB , relA ' ) ΔmepM was performed with pHV30 vector or recombinant plasmids encoding each of eight endopeptidases under control of PrhaBAD promoter of TIS1 translation initiation signal . Induction of endopeptidase ( ED ) genes was performed with 0 . 2 % L - rhamnose in presence or absence of 8 µg / ml ceftriaxone . BHI agar plates contained 40 µM IPTG 1 % L - arabinose for induction of ycbB relA ' , respectively .

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Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.


( B ) plating efficiency was performed with derivatives of BW25113 Δ8EDs pHV53 ( mepM ) harboring vector pHV7 or recombinant plasmids carrying each of eight endopeptidase genes under control of ParaBAD promoter . In this , functional replacement of MepM is detected on growth in media containing 0 . 2 % or 1 % L - arabinose for expression of endopeptidase gene carried by vector pHV7 , while by - passing requirement for induction of mepM copy of pHV53 by L - rhamnose . Complementation was observed with both concentrations of inducer for mepH , mepS , pbpG . Overproduction of mepM encoded by pHV7 derivative in presence of high dose of L - arabinose ( 1 % ) was lethal . right panel presents growth control performed in presence of 0 . 2 % L - rhamnose for induction of mepM copy carried by pHV53 .

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(B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.


( B ) Functional complementation of mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with pHV30 vector or recombinant plasmids encoding each of eight endopeptidases under control of PrhaBAD promoter . Induction of ycbB of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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(B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Functional complementation of mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with pHV30 vector or recombinant plasmids encoding mepA , mepK , mepS or derivatives encoding catalytically inactive endopeptidases under control of PrhaBAD promoter . ( B ) complementation was repeated for BW25113 Δ7EDs pHV63 ( ycbB ) , which was obtained by deletion of chromosomal endopeptidase genes except mepM . Induction of ycbB of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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(A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Growth curves of ( i ) BW25113 ( ycbB , relA ' ) its ΔsltY derivative ; ( ii ) M1 its derivatives ( M1 . 1 to 1 . 4 ) harboring mutations in sltY . growth medium contained ( i ) 40 µM IPTG , 1 % L - arabinose , 8 µg / ml ceftriaxone for BW25113 ( ycbB , relA ' ) its ΔsltY derivative or ( ii ) 50 µM IPTG 16 µg / ml ampicillin for M1 its derivatives .

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(A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives.


A . Intracellular survival of strains 188 , 188SLO - ( SLO - ) , 188NADase - ( NADase - ) . Intracellular CFU were quantified at 2 h , 4 h , 6 h , 8 h , 24 h , intracellular survival was calculated as viable CFU compared to 2 h .

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A. Intracellular survival of GAS strains 188, 188SLO- (SLO-), and 188NADase- (NADase-). Intracellular CFU were quantified at 2 h, 4 h, 6 h, 8 h, and 24 h, and intracellular survival was calculated as the percent viable CFU compared to 2 h.


B . Intracellular survival of 188SLO - that was partially complemented from plasmid piSLO ( SLO - ( piSLO ) ) , 188 188SLO - that contained empty complementation vector , 188 ( pSIV4 ) SLO - ( pSIV4 ) , respectively .

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B. Intracellular survival of GAS strain 188SLO- that was partially complemented from plasmid piSLO (SLO-(piSLO)), strain 188 and 188SLO- that contained the empty complementation vector, 188(pSIV4) and SLO-(pSIV4), respectively.


C . Intracellular survival of JRS4 , JRS4SLO - ( SLO - ) , JRS4NADase - ( NADase - ) . Experiments were performed in triplicate values represent mean of three independent experiments ± SD . * , P < 0 . 001 .

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C. Intracellular survival of GAS strain JRS4, JRS4SLO- (SLO-), and JRS4NADase- (NADase-). Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD. *, P<0.001.


A . Confocal microscopy of association between EGFP - LC3 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 Alexa - 660 , red blue , respectively ) . Scale bar = 10 µm . of intracellular that were associated with EGFP - LC3 at 3 h post - infection is shown for each .

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A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain.


B . Electron microscopy of strains 188 , 188SLO - , 188SLO - SLS - at 3 h post - infection . Arrowheads indicate presence of double or multiple membranes ( 188 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding . Vacuolar compartments associated with 188 188SLO - also contain material .

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B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material.


C . Intracellular survival of strains 188 , 188SLO - , 188SLS - , 188SLO - SLS - . * , P < 0 . 05 .

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C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05.


D . Intracellular survival of 188 or 188SLO - in presence or absence of Beclin1 knockdown .

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D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.


A . Intracellular survival of strains 188 , 188NADase - , 188NADase ( G330D ) . Deletion of gene encoding NADase or inactivation of NADase by acid substitution G330D resulted in a 20 - to 40 - fold reduction in intracellular survival compared to parent 188 . * , P < 0 . 001 .

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A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001.


activity measurements ( C ) of culture supernatants from strains 188 , 188NADase - , 188NADase ( G330D ) .

and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).


A . Confocal microscopy demonstrating association between ubiquitin ( green ) intracellular ( red ) at 30 min post - infection in keratinocytes infected with 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular were stained blue red for identification as described in Figure 2 . Scale bar = 10 µm . of intracellular that were associated with ubiquitin is indicated for each , on of at least 100 in three independent experiments .

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A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments.


B . Hemolytic activity of culture supernatants of 188 , 188SLO - , 188SLO ( Y255A ) . acid substitution Y255A in SLO abrogates ability of SLO to porate host , demonstrated by failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of variant SLO . Inset , corresponding Western blot for SLO in culture supernatants from these strains .

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B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains.


. C . NADase activity in culture supernatants in cytosol of keratinocytes infected with 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular was identical between 771 771SLO ( Y255A ) , but reduced in 771SLO - .

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. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-.


D . intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental , 188 , similar to that of 188SLO - . * , P < 0 . 01 .

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D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01.


E . Confocal microscopy demonstrating association between galectin 8 ( green ) intracellular ( red ) in keratinocytes infected for 30 min with 188 , 188SLO - , or 188 SLO - SLS - . Extracellular ( ( out ) ) were stained blue red for identification as described in Figure 2 . Scale bar = 10 µm . of intracellular that were associated with galectin 8 is indicated for each , on of at least 100 in three independent experiments . 188SLO - SLS - was never associated with galectin 8 ( 0 % ) .

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E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).


SLO NADase inhibit lysosomal fusion to - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with 188 , 188SLO - , or 188NADase - demonstrating association of lysosomal marker LAMP - 1 ( red ) with ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , 6 h post - infection . Scale bar = 10 µm . B . of of within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular were quantified for each time point in each experiment .

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SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.


( B ) Representative immunoblot of Nrxn1β - Fc variants harvested from HEK293 media subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE immunoblotting with antibodies against Fc - tag 3G10 epitope ( to detect HS stub which reveal HS after heparinase digestion ) . ( C ) of 3G10 ( reflecting HS ) signal normalized to that of Fc ( Nrxn1 ) . Bar graph shows means ± of 3 independent experiments .

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(B) Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against the Fc-tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). (C) Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ±SEM of 3 independent experiments.


HEK293 : ( B ) Representative immunoblots ( of three independent experiments ) with antibodies against HA , 3G10 epitope ( for HS stub ) V5 .

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HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5.


HEK293 : ( D ) Representative immunoblots ( of three independent experiments ) analyzed as described for ( B ) .

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HEK293 cells: (D) Representative immunoblots (of three independent experiments) analyzed as described for (B).


( F - H ) cortical neurons : Cortical neurons were infected with control lentivirus ( empty vector ) or lentivirus expressing FLAG - tagged CA10 , lysates subjected to immunoprecipitation for neurexin heparinase treatment . ( F ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 ( for HS stub ) FLAG ( for CA10 ) . Stars indicate non - neurexin bands ( see results ) . ( G ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total neurexin amounts [ upper band ( ) + LMW ] . Data shown as mean ± ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak . ( H ) of 3G10 signal normalized to signal of corresponding neurexin band . Data shown as mean ± of biological replicates . * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak ( n = 3 ) .

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(F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3).


( I - K ) Mouse brains : Neurexins CA10 were immunoprecipitated from total mouse brain lysates subject to heparinase treatment . ( I ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 epitope ( for HS stub ) CA10 . Stars indicate non - neurexin bands ( see results ) . ( J ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total neurexin amounts [ upper band ( ) + LMW ] . Data shown as mean ± ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak . ( K ) of 3G10 signal normalized to signal of corresponding neurexin band . Data shown as mean ± of 3 biological replicates . * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak .

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(I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.


( B ) Representative immunoblot of indicated harvested from HEK293 media subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE immunoblotting with antibodies against Fc ( for Nrxn1β ) , 3G10 epitope ( for HS stub ) V5 ( for CA10 ) . Input samples were analyzed separately for CA10 to ensure equal expression ( lower panel ) . ( C ) of 3G10 epitope signal , normalized to Fc ( Nrxn1β ) . Data shown are mean ± of 3 independent replicates . * , p < 0 . 05 ; by Mann - Whitney comparing each Nrxn1 mutant with without CA10 ( n = 3 ) . No significance difference was observed for S & T > G ( p = 0 . 1143 ) , GIGG ( p = 0 . 9714 ) or DIRR ( p = 0 . 4857 ) variants .

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(B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel). (C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants.


( E ) representative immunoblots analyzed using antibodies against Fc - tag ( for Nrxn1β - Fc ) 3G10 epitope ( for HS stub ) V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . ( F , of 3G10 epitope signal normalized to Fc ( for Nrxn1β - Fc ) signals , Data shown are mean ± of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak .

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(E) representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) signals, Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


( G ) show representative immunoblots analyzed using antibodies against HA tag ( Nrxn1α - HA ) , 3G10 epitope ( for HS stub ) V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . H ) of 3G10 epitope signal normalized to HA ( for Nrxn1α - HA ) signals , respectively . Data shown are mean ± of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak .

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(G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


( D ) Comparison of 1H - 13C HSQC spectra of Nrxn1 stalk ( red ) Nrxn1 stalk / CA10 ( black ) at a 2 : 1 molar ( 50 mM phosphate buffer , pH 7 . 4 , 0 . 5 mM d10 - DTT , 10 % D2O ) . Nrxn1 residues with stronger chemical shift changes are indicated blue boxes . Asterisk ( * ) indicates an impurity from glycerol .

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(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.


( B - C ) Extracted ion chromatograms ( EIC ) of DDILVASAECPSDDE peptides its glycosylated forms , from Nrxn1β expressed in HEK293 .

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(B-C) Extracted ion chromatograms (EIC) of DDILVASAECPSDDE peptides and its glycosylated forms, from Nrxn1β expressed in HEK293 cells.


( D ) , expressed as % of total occupancy of each glycosylation form , upon control CA10 expression conditions . Bar graph shows means ± of 4 biological replicates ; * , p < 0 . 05 by Mann - Whitney comparing occupancy of each glycosylation with without CA10 ( n = 4 biological replicates ) .

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(D) Quantification, expressed as % of total occupancy of each glycosylation form, upon control and CA10 expression conditions. Bar graph shows means ±SEM of 4 biological replicates; *, p<0.05 by Mann-Whitney tests comparing the occupancy of each glycosylation with and without CA10 (n=4 biological replicates).


( A ) Immunoblot α - showing neurexin isoform distribution in cortex , hippocampus , midbrain cerebellum dissected from 8 - week - old mice . ( B ) of lower molecular neurexin ( LMW ; arrowhead ) in relation to total of α - neurexins [ upper band ( ) + LMW ] . Bar graphs show mean ± of 3 independent replicates * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak , comparing to in cerebellum .

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(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice. (B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum.


( C ) α - Neurexin immunoprecipitated from forebrain ( Fb ) cerebellum ( Cb ) using an anti - pan - neurexin antibody was heparinase - treated ( ' Heps ' ) analyzed by immunoblotting with antibodies against neurexin 3G10 monoclonal ( to detect HS stub ) . Stars indicate non - neurexin bands ( see results ) . ( D ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total of α - neurexins [ upper band ( ) + LMW ] . Bar graphs show mean ± SEMof 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak comparing forebrain cerebellum . ( E ) of 3G10 signal normalized to signal of corresponding neurexin band . Bar graphs show mean ± of 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak comparing forebrain cerebellum .

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(C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.


( A - B ) surface binding to endogenous neurexins . ( A ) Recombinant Fc - tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction . Surface - bound was quantified as Fc signal of normalized to a for endogenous neurexin ( n = 4 cultures ; three fields imaged averaged per well ) . Scatchard for LRRTM2 revealed an apparent Kd of 29 . 5 nM a Bmax of 0 . 49 nM for control Kd 35 . 3 nM , Bmax 0 . 28 nM upon expression of CA10 ; significant ( p = 0 . 0006 ) by non - linear regression . Data shown as mean ± of biological replicates ( n = 4 ) . ( B ) Increasing amounts of recombinant , Fc - tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction , analyzed as in ( A ) . apparent Kd for Nlgn1 was 186 nM Bmax 1 . 65 nM for control Kd 223 nM , Bmax 1 . 49 nM when CA10 was overexpressed . difference was not significant ( p = 0 . 20 ) by non - linear regression . Data shown as mean ± of biological replicates ( n = 4 ) .

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(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4).