A . Raw 264 . 7 macrophage were transfected with FLAG ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker ) , or mkate2 ( N - term ) - 2xTAPP1 - PH domain ( PI ( 3 , 4 ) P2 marker ) . Representative photomicrographs ( from 25 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . bounding boxes show " High Mag " panels that magnify representative vesicles . colocalization of Rab10 indicated markers in bounding boxes were analyzed using Image J as described in method shown in " CoLoc " panels . colocalized pixels are shown in with same . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate representative Rab10 vesicles positively colocalized with indicated marker . B . At least 25 across three independent experiments were analyzed for each condition ( see Materials Methods section ) . % of Rab10 vesicles positive with indicated marker within each was quantified . Each dot represents mean value from one analyzed . Group means are given , with error bars representing ± .

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

A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM.


C . Raw 264 . 7 macrophage were transfected with FLAG ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) treated with wortmannin ( 1 μM ) for one hour . Representative photomicrographs ( from 25 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . Scale bars are 10 μm , or 1 μm in " Hig Mag " panel . D . of treated with or without wortmannin harboring Rab10 vesicles was calculated from > 30 across five independent experiments . Each dot on plot represents mean values from one experiment . Error bars represent ± significance was assessed by Mann - Whitney ( owing to non - normal distributions ) with * * indicating P < 0 . 01 .

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C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01.


E . Raw 264 . 7 were co - transfected with eGFP ( N - term ) - Rab10 mRuby ( N - term ) - Rab5 for 48 hours , with live - recordings for ~ 10 minutes . A representative vesicle is shown over presented time - lapse . Scale bar represents 1 μm . F . intensities of vesicles are calculated over time . Dots show mean values calculated from n = 8 recorded vesicles from from n = 3 biologically independent experiments . Error bars show ± .

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E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm. F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , were incubated with ( A ) TRITC - dextran ( 70 kDa , shown as red signal ) Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , were incubated with ( B ) DQ - ovalbumin , for 30 min before washing fixation . Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . ( C , D ) were further stained for ( C ) EEA1 , or ( D ) Lamp1 , which was detected with Cy5 dye ( show as red signal ) . Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


E ) 25 across three independent experiments were analyzed for each condition . % of Rab10 vesicles positive with indicated marker in each were quantified . Each dot represents mean results from vesicles analyze in one . group means of % Rab10 vesicles positive with marker are shown , with error bars representing ± . Significance was assessed by Mann - Whitney for each marker , where * * representing p < 0 . 01 , * * * * p < 0 . 0001 N . S p > 0 . 05 .

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E) 25 cells across three independent experiments were analyzed for each condition. % of Rab10 vesicles positive with the indicated marker in each cell were quantified. Each dot represents the mean results from vesicles analyze in one cell. The group means of % Rab10 vesicles positive with the marker are shown, with error bars representing ± SEM. Significance was assessed by Mann-Whitney test for each marker, where ** representing p<0.01, **** p<0.0001 and N.S p>0.05.


A . Raw 264 . 7 macrophage were transfected with Flag ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker , epifluorescence , shown in blue ) followed by anti - Flag immunofluorescence ( shown in green ) pT73 - Rab10 antibody ( shown in red ) . To evaluate colocalization of phospho - Rab10 with early endosomes or late endosome / lysosome markers , immunofluorescence detection was performed for endogenous EEA1 or Lamp1 ( shown in blue ) . Raw 264 . 7 macrophage were further fed 0 . 05 mg mL - 1 of 70 kDa TRITC - conjugated dextran ( epifluorescence , shown in blue ) or 0 . 05 mg mL - 1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence . Representative were selected from 25 analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc ( pRab10 / Rab10 ) ' ' CoLoc ( pRab10 / Marker ) ' columns are indicated . arrow heads highlight vesicles scored as co - positive between pT73 - Rab10 Rab10 or indicated marker . B . 25 across three independent experiments were analyzed for each condition , with each dot representing mean of vesicle across . group means of % pT73 - Rab10 vesicles positive for indicated marker are given , with error bars showing ± .

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A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in the "CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker. B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM.


A . Raw264 . 7 were transfected with Flag ( N - term ) - LRRK2 eGFP ( N - term ) - Rab10 for 24 hours treated with or without LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from > 20 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) , immuno - stained for pT73 - Rab10 ( shown as red signal ) LRRK2 ( shown as blue signal ) . bounding boxes are magnified in " High Mag " panels that show vesicles . Scale bars represent 10 μm or 1 μm in " Hig Mag " panels .

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A. Raw264.7 cells were transfected with Flag(N-term)-LRRK2 and eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells with eGFP(N-term)-Rab10 epifluorescence (shown as green signal), and immuno-stained for pT73-Rab10 (shown as red signal) and LRRK2 (shown as blue signal). White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm or 1 μm in "Hig Mag" panels.


B . Raw 264 . 7 macrophage transfected with eGFP ( N - term ) - Rab10 Flag ( N - term ) - LRRK2 were sequentially lysed mechanically into buffer to create a ' soluble ' fraction , then insoluble material lysed into triton X - 100 buffer to create a " triton x - 100 " fraction . " soluble " triton X - 100 buffer solubilized fractions were analyzed using SDS - PAGE ( TGX gels ) , representative immunoblots from n = 3 independent experiments are shown .

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B. Raw 264.7 macrophage cells transfected with eGFP(N-term)-Rab10 and Flag(N-term)-LRRK2 were sequentially lysed mechanically into buffer to create a 'soluble' protein fraction, and then insoluble material lysed into triton X-100 buffer to create a "triton x-100" fraction. The "soluble" and triton X-100 buffer solubilized fractions were analyzed using SDS-PAGE (TGX gels), and representative immunoblots from n=3 independent experiments are shown.


C . " triton X - 100 " fraction was used as input for LRRK2 immunoprecipitation , with subsequent detection of total Rab10 pT73 - Rab10 in LRRK2 : Rab10 immunocomplexes using SDS - PAGE ( TGX gels ) . To measure stoichiometry of pT73 - Rab10 to total Rab10 , same immunocomplexes were analyzed using phos - tag SDS - PAGE method , with unphosphorylated eGFP ( N - term ) - Rab10 migrating below phospho - Rab10 . Immunoblots are representative of three independent experiments , with similar results obtained .

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C. The "triton X-100" fraction was used as input for LRRK2 immunoprecipitation, with subsequent detection of total Rab10 and pT73-Rab10 proteins in LRRK2:Rab10 immunocomplexes using SDS-PAGE (TGX gels). To measure the stoichiometry of pT73-Rab10 to total Rab10, the same immunocomplexes were analyzed using phos-tag SDS-PAGE method, with unphosphorylated eGFP(N-term)-Rab10 migrating below the phospho-Rab10 protein. Immunoblots are representative of three independent experiments, with similar results obtained.


D . Calculated of total LRRK2 : Rab10 with without LRRK2 kinase inhibition . Data are from n = 3 biologically independent experiments with error bars showing ± . Significance was assessed by a two - t - with N . S representing P > 0 . 05 .

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D. Calculated levels of total LRRK2:Rab10 protein complex with and without LRRK2 kinase inhibition. Data are from n=3 biologically independent experiments with error bars showing ± SEM. Significance was assessed by a two-tailed t-test with N.S representing P>0.05.


E . signal intensities from pT73 - Rab10 unphosphorylated Rab10 in input lysate co - immunoprecipitated with LRRK2 antibodies . stoichiometry of pT73 - Rab10 was calculated by dividing pT73 - Rab10 signal by total Rab10 signal ( total Rab10 signal equivalent to pT73 - Rab10 signal + unphosphorylated Rab10 signal ) . Data are from n = 3 biologically independent experiments with error bars showing ± ; Significance was assessed by two - t - with * * * representing P < 0 . 0005 .

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E. The signal intensities from pT73-Rab10 and unphosphorylated Rab10 in the input lysate co-immunoprecipitated with LRRK2 antibodies. The stoichiometry of pT73-Rab10 was calculated by dividing the pT73-Rab10 signal by total Rab10 signal (total Rab10 signal equivalent to pT73-Rab10 signal+ unphosphorylated Rab10 signal). Data are from n=3 biologically independent experiments with error bars showing ± SEM; Significance was assessed by two-tailed t-test with *** representing P<0.0005.


A , B . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - LRRK2 mBAC transgenic mice were pre - incubated with FITC - conjugated dextran ( 70 kDa , green signal ) prior to washing , fixing , immunostaining with total - Rab10 ( shown as red signal ) , or pT73 - Rab10 antibody ( shown as red signal ) . Alternatively , were co - stained with antibodies to Rab5 , Rab7 or Lamp1 ( shown as green signal in separate ) . Representative photomicrographs ( from > 30 analyzed for each condition from n = 3 biologically independent experiments ) are shown . bounding boxes are " High Mag " panels that show vesicles analyzed . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 indicated marker . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels .

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A,B. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were pre-incubated with FITC-conjugated dextran (70 kDa, green signal) prior to washing, fixing, and immunostaining with total-Rab10 (shown as red signal), or pT73-Rab10 antibody (shown as red signal). Alternatively, cells were co-stained with antibodies to Rab5, Rab7 or Lamp1 (shown as green signal in separate images). Representative photomicrographs (from >30 images analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels.


C . of 20 across three independent experiments are given for each indicated co - localization , where % of Rab10 or pT73 - Rab10 vesicles positive with indicated markers was calculated for each ( with signal inside of vesicles analyzed for TRITC - dextran DQ - ovalbumin ) . Each dot represents mean value of vesicles analyzed inside one . group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated . Error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * * * * representing p < 0 . 0001 N . S representing p > 0 . 05 .

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C. Analysis of 20 cells across three independent experiments are given for each indicated co-localization, where % of Rab10 or pT73-Rab10 vesicles positive with indicated markers was calculated for each cell (with signal inside of vesicles analyzed for TRITC-dextran and DQ-ovalbumin). Each dot represents the mean value of vesicles analyzed inside one cell. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated. Error bars represent ± SEM. Significance was assessed by Mann-Whitney tests for each marker with **** representing p<0.0001 and N.S representing p>0.05.


D . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - LRRK2 mBAC transgenic mice were treated with 0 . 001 % DMSO or 100 nM MLi2 ( i . e . , IC90 ) for two hours prior to immunostaining with total Rab10 or pT73 - Rab10 antibody ( shown as red signal ) , anti - Rab5 or anti - Rab7antibody ( shown as green signal ) . Representative photomicrographs ( from 20 analyzed for each condition from n = 3 biologically independent experiments ) are shown . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 indicated marker .

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D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were treated with 0.001% DMSO or 100 nM MLi2 (i.e., IC90 concentration) for two hours prior to immunostaining with total Rab10 or pT73-Rab10 antibody (shown as red signal), and anti-Rab5 or anti-Rab7antibody (shown as green signal). Representative photomicrographs (from 20 cells analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker.


E . Calculated of double - positive vesicles ( with > 50 % of vesicle boundary demarcated by signal in colocalization ) from 20 counted from n = 3 biologically independent experiments are shown . Each dot represents 1 in dotted plot . group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated shown . Error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * representing p < 0 . 05 , * * P < 0 . 01 .

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E. Calculated percentage of double-positive vesicles (with >50% of the vesicle boundary demarcated by white signal in the colocalization analysis) from 20 cells counted from n=3 biologically independent experiments are shown. Each dot represents 1 cell in the dotted plot. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated and shown. Error bars represent± SEM. Significance was assessed by Mann-Whitney test for each marker with * representing p<0.05, ** P<0.01.


F . Raw 264 . 7 macrophage were transfected with eGFP ( N - term ) - Rab10 for 24 hours treated with or without LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from 25 analyzed for each condition across n = 3 biologically independent experiments ) are shown with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) pT73 - Rab10 ( shown as red signal ) , with EEA1 ( shown as blue signal ) . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 Rab10 indicated marker . G . Calculated of colocalized vesicles , with each dot representing mean vesicle frequency in each analyzed . group mean of % Rab10 vesicles positive with EEA1 were calculated , error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * * * representing P < 0 . 005 .

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F. Raw 264.7 macrophage cells were transfected with eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from 25 images analyzed for each condition across n=3 biologically independent experiments) are shown with eGFP(N-term)-Rab10 epifluorescence (shown as green signal) and pT73-Rab10 (shown as red signal), with EEA1 (shown as blue signal). White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 Rab10 and the indicated marker. G. Calculated percentage of colocalized vesicles, with each dot representing mean vesicle frequency in each cell analyzed. The group mean of % Rab10 vesicles positive with EEA1 were calculated, and error bars represent ± SEM. Significance was assessed by Mann-Whitney test for each marker with *** representing P<0.005.


A . Raw264 . 7 were transfected with Flag - Rab10 or control empty vector . transfected were lysed in presence of 2 mM GTPγS or 2 mM GDP . Flag - Rab10 was immunoprecipitated with Flag - resin followed by extensive washing elution of complexes with 200 μg mL - 1 3xFlag - tide . Eluates were analyzed by SDS - PAGE followed by immuno - blotting with anti - Flag - HRP antibody .

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A. Raw264.7 cells were transfected with Flag-Rab10 or control empty vector. The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP. The Flag-Rab10 complex was immunoprecipitated with Flag-resin followed by extensive washing and elution of complexes with 200 μg mL-1 3xFlag-tide. Eluates were analyzed by SDS-PAGE followed by immuno-blotting with anti-Flag-HRP antibody.


C . Flag ( N - term ) - Rab10 was purified from HEK293 - FT transfected with Flag ( N - term ) - Rab10 plasmids immobilized on Flag - resin in lysis buffer containing 2 mM GTPγS , or 2 mM GDP , as indicated . Beads were mixed with lysates from transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to generate Rab10 : EHBP1L1 complexes . Total Rab10 on beads was detected using anti - Flag antibody , EHBP1L1 immobilized to total Rab10 detected with an anti - Myc antibody . Representative immunoblots from n = 3 biologically independent experiments are shown . D . of relative EHBP1L1 pulled down . Data are from n = 3 biologically independent experiments . Error bars represent ± . . .

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C. Flag(N-term)-Rab10 protein was purified from HEK293-FT cells transfected with Flag(N-term)-Rab10 plasmids and immobilized on Flag-resin in lysis buffer containing 2 mM GTPγS, or 2 mM GDP, as indicated. Beads were mixed with lysates from cells transfected with Myc-mkate2(N-term)-EHBP1L1 to generate Rab10:EHBP1L1 complexes. Total Rab10 on the beads was detected using anti-Flag antibody, and EHBP1L1 immobilized to total Rab10 detected with an anti-Myc antibody. Representative immunoblots from n=3 biologically independent experiments are shown. D. Quantification of relative EHBP1L1 pulled down. Data are from n=3 biologically independent experiments. Error bars represent ± SEM...


E . HEK293 - FT were transfected with Flag ( N - term ) - Rab10 Myc ( N - term ) - LRRK2R1441C to induce Rab10 phosphorylation . bound Flag ( N - term ) - Rab10 was extracted with Flag - resin to generate ' pRab10 beads , ' or extracted from treated with MLi2 ( 100 nM ) to generate ' Rab10 beads . ' Beads were mixed with lysates from HEK293 - FT transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to form on - bead Rab10 : EHBP1L1 complexes . total Rab10 pT73 - Rab10 on beads was detected using anti - Flag antibody pT73 - Rab10 antibody . EHBP1L1 in lysates immobilized on beads was detected using anti - Myc tag antibody . Representative immunoblots are shown . F . relative EHBP1L1 pulled down , normalized to total Rab10 on - bead , was determined from n = 3 biologically independent experiments . Error bars represent ± . Each dot represents one experiment , with significance assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc P < 0 . 0001 .

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E. HEK293-FT cells were transfected with Flag(N-term)-Rab10 and Myc(N-term)-LRRK2R1441C to induce Rab10 phosphorylation. Membrane bound Flag(N-term)-Rab10 protein was extracted with Flag-resin to generate 'pRab10 beads,' or extracted from cells treated with MLi2 (100 nM) to generate 'Rab10 beads.' Beads were mixed with lysates from HEK293-FT cells transfected with Myc-mkate2(N-term)-EHBP1L1 to form on-bead Rab10:EHBP1L1 complexes. The total Rab10 and pT73-Rab10 on the beads was detected using anti-Flag antibody and pT73-Rab10 antibody. The EHBP1L1 in the lysates and immobilized on the beads was detected using anti-Myc tag antibody. Representative immunoblots are shown. F. The relative EHBP1L1 pulled down, normalized to total Rab10 protein on-bead, was determined from n=3 biologically independent experiments. Error bars represent ± SEM. Each dot represents one experiment, with significance assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001.


G . Raw 264 . 7 were co - transfected with eGFP ( N - term ) - Rab10 ( epifluorescence shown in green ) Myc - mkate2 ( N - term ) - EHBP1L1 ( epifluorescence shown in blue ) , or Flag ( C - term ) - RILPL2 ( anti - Flag signal shown in blue ) . were stained for pT73 - Rab10 ( shown in red ) . Representative photomicrographs of vesicles ( from > 10 analyzed for each condition from n = 3 biologically independent experiments ) are shown . Scale bar represents 1 μm . H . Pearson ' s colocalization coefficient for Rab10 pT73 - Rab10 , EHBP1L1 RILPL2 on vesicle bodies tubular vesicles . Data are from n = 20 vesicles with tubular structures positive for pT73 - Rab10 , n = 12 vesicles with tubular structures positive for EHBP1L1 , or RILPL2 , calculated from from three independent experiments . Each dot represents one vesicle or tubular structure , with significance assessed by Kruskal - Wallis followed by Dunn ' s multiple comparison with * * * representing P < 0 . 0005 * * * * P < 0 . 0001 . Error bars represent ± . .

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G. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence shown in green) and Myc-mkate2(N-term)-EHBP1L1 (epifluorescence shown in blue), or Flag(C-term)-RILPL2 (anti-Flag signal shown in blue). Cells were stained for pT73-Rab10 (shown in red). Representative photomicrographs of individual vesicles (from >10 cells analyzed for each condition from n=3 biologically independent experiments) are shown. Scale bar represents 1 μm. H. Pearson's colocalization coefficient for Rab10 and pT73-Rab10, EHBP1L1 and RILPL2 on vesicle bodies and tubular vesicles. Data are from n=20 vesicles with tubular structures positive for pT73-Rab10, and n=12 vesicles with tubular structures positive for EHBP1L1, or RILPL2, calculated from cells from three independent experiments. Each dot represents one vesicle body or tubular structure, with significance assessed by Kruskal-Wallis test followed by Dunn's multiple comparison test with *** representing P<0.0005 and **** P<0.0001. Error bars represent ± SEM..


I . Raw264 . 7 were co - transfected with Flag ( N - term ) - Rab10 , Flag ( N - term ) - LRRK2R1441C , Myc ( N - term ) - EHBP1L1 , with representative immunoblots shown . J . Relative pT73 - Rab10 were calculated as fold of transfected with Flag ( N - term ) - Rab10 Flag ( N - term ) - LRRK2 only . Data are from n = 3 biologically independent experiments . Each dot represents one independent experiment . Significance was assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc P < 0 . 0001 .

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I. Raw264.7 cells were co-transfected with Flag(N-term)-Rab10, Flag(N-term)-LRRK2R1441C, and Myc(N-term)-EHBP1L1, with representative immunoblots shown. J. Relative pT73-Rab10 levels were calculated as fold of cells transfected with Flag(N-term)-Rab10 and Flag(N-term)-LRRK2 only. Data are from n=3 biologically independent experiments. Each dot represents one independent experiment. Significance was assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001.


K . Raw264 . 7 were co - transfected with eGFP ( N - term ) - Rab10 ( epifluorescence , shown in green ) Myc - mkate2 ( N - term ) - EHBP1L1 or Myc - mkate2 control plasmid ( epifluorescence , shown in red ) . Representative photomicrographs are shown . Bounding boxes highlight vesicles in " High Mag " panels . Scale bars represent 10 μm , 1 μm for " High Mag " . L . % of eGFP ( N - term ) - Rab10 vesicles positive with pT73 - Rab10 were calculated ( from 25 analyzed for each condition from n = 3 biologically independent experiments ) . Each dot represents mean frequency observed in one . Error bars show ± with significance was assessed by Mann - Whitney . * * * represents P < 0 . 0005 .

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K. Raw264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence, shown in green) and Myc-mkate2(N-term)-EHBP1L1 or Myc-mkate2 control plasmid (epifluorescence, shown in red). Representative photomicrographs are shown. Bounding boxes highlight vesicles in "High Mag" panels. Scale bars represent 10 μm, and 1 μm for "High Mag" images. L. % of eGFP(N-term)-Rab10 vesicles positive with pT73-Rab10 were calculated (from 25 cells analyzed for each condition from n=3 biologically independent experiments). Each dot represents the mean frequency observed in one cell. Error bars show ± SEM with significance was assessed by Mann-Whitney test. *** represents P<0.0005.


A - D . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - mLRRK2 BAC transgenic mice were immunostained with anti - mouse CCR5 , or CD11b , or MHC II antibody ( shown as green signal ) ( A ) total Rab10 antibody ( red signal ) , or ( B ) pT73 - Rab10 antibody ( red signal ) as indicated . Representative photomicrographs ( from > 20 analyzed for each condition from n = 3 biologically independent experiments ) are shown . ( C ) Photomicrographs showing different cargo with respect to Rab10 tubular structures . ( D ) EEA1 ( shown as green signal ) with total Rab10 or pT73 - Rab10 ( shown as red signals ) as indicated . Bounding boxes in panels A - D are magnified in " High Mag " panels that show vesicles . Scale bars represent 10 μm 2 μm for " High Mag " .

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A-D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were immunostained with anti-mouse CCR5, or CD11b, or MHC II antibody (shown as green signal) and (A) total Rab10 antibody (red signal), or (B) pT73-Rab10 antibody (red signal) as indicated. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown. (C) Photomicrographs showing the different cargo with respect to Rab10 tubular structures. (D) EEA1 (shown as green signal) with total Rab10 or pT73-Rab10 (shown as red signals) as indicated. Bounding boxes in panels A-D are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm and 2 μm for "High Mag" images.


E . Calculated of vesicles positive with tubular structures ( n > 30 vesicles analyzed from from n = 3 biologically independent experiments ) are shown . Each dot represents one vesicle , with significance assessed by Mann - Whitney . * * represents P < 0 . 01 . Error bars represent ± .

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E. Calculated percentage of vesicles positive with tubular structures (n>30 vesicles analyzed from cells from n=3 biologically independent experiments) are shown. Each dot represents one vesicle, with significance assessed by Mann-Whitney test. ** represents P<0.01. Error bars represent ± SEM.


F . BMDM were treated with 0 . 004 % DMSO or 100 nM MLi2 prior to live surface with Alexa - 594 conjugated rat anti - mouse CD11b antibody on ice for ten minutes . Three wells of from each condition were fixed imaged for initial surface CD11b binding ( shown in red as indicated ) . Six wells of from each condition were further incubated at 37 ° C to allow rapid internalization of CD11b - antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0 . 5 % acetic acid on ice for one min . Three of six wells were fixed surface - stained with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes remaining on surface ( shown in green as indicated ) . Internalized CD11b - antibody complexes are shown in red . other three wells of were further incubated at 37 ° C again for ten min to allow recycling of internalized CD11b - antibody followed by surface with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes recycled back to surface . Representative from n = 3 biologically independent experiments are shown . Scale bars represent 30 μm . G - I . ( G ) initial surface CD11b , or ( H ) internalized CD11b , or ( I ) recycled CD11b were calculated from > 20 analyzed for each condition from n = 3 biologically independent experiments . Each dot represents mean value from one experiment . Error bars show ± with significance assessed by Mann - Whitney . * represents P < 0 . 05 .

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F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05.


A . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - mLRRK2 BAC transgenic mice were treated with DMSO , 1 μM Rab10 anti - sense oligonucleotide for 4 days , 100 nM MLi2 for 12 hours , as indicated , before stimulation with 100 nM CCL5 for indicated time ( hours ) . Representative immunoblots from n = 3 biologically independent experiments are shown . B . Relative pSer473 - Akt were normalized to total Akt calculated as fold of naïve ( without CCL5 stimulation ) . Data shown are group means ± from n = 3 biologically independent replicates , with significance assessed by one - way ANOVA with Tukey ' s post hoc with treated groups compared to matched control groups , with * * representing P < 0 . 01 .

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A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown. B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01.


C . BMDM , as described in panel ( A ) , were additionally treated with DMSO ( control ) , EIPA , or Akti - 1 / 2 ( Akt1 / 2 inhibitor ) as indicated , for 1 hour after CCL5 stimulation . Representative immunoblots from n = 3 biologically independent experiments are shown . D . Relative pSer473 - Akt were normalized to total Akt calculated as fold of naïve ( without CCL5 stimulation ) . Each dot represents one experiment , with error bars showing ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * representing P < 0 . 0005 , * * * * P < 0 . 0001 .

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C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001.


E . BMDM , as described in panels ( A C ) , were seeded in Transwell ( Sigma ) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers . After two hours , failing to migrate through matrices were removed with a cotton swab . Migrated were counted with DAPI , with representative shown at low magnification . Representative from n = 3 biologically independent experiments are shown . F . Relative chemotaxis activity was calculated as fold of naïve that migrated without any added CCL5 . Each dot represents mean of collected from one experiment . Data are group means ± , with significance assessed by multiple t . , with * representing P < 0 . 05 .

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E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown. F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05.


( A ) Mean clinical scores ( ± ) of C57BL / 6 → C57BL / 6 controls treated with DTX CD11c - DTR / GFP → C57BL / 6 treated with DTX or PBS during whole observation period starting from day - 1 . received in vitro - generated 2d2 . tdRFP Th17 intravenously on day 0 . DC depletion in CD11c - DTR / GFP → C57BL / 6 reduces encephalitogenicity of adoptively transferred 2d2 . tdRFP Th17 . Mann - Whitney - U - was performed on mean clinical scores ( n = 11PBS / 13DTX ) ; * p < 0 . 05 , * * p < 0 . 01 ; see also Appendix Table S1 . Pooled data from two independent experiments .

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(A) Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day -1. All animals received in vitro-generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17. Mann-Whitney-U-test was performed on mean clinical scores (n=11PBS/13DTX); * p < 0.05, ** p < 0.01; see also Appendix Table S1. Pooled data from two independent experiments.


( B ) Mononuclear were isolated on day 30 of shown in ( A ) from CNS of PBS - DTX - treated CD11c - DTR / GFP → C57BL / 6 that had been transferred with 2d2 . tdRFP Th17 . Flow cytometry was performed on surface stained samples . were pre - gated on lymphocyte ( FSC SSC gate ) PI - negative ; pooled data of three ; representative of two independent experiments .

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(B) Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17. Flow cytometry was performed on surface stained cell samples. Cells were pre-gated on lymphocyte cells (FSC SSC gate) and PI-negative; pooled data of three animals; representative of two independent experiments.


( C ) Mononuclear were isolated from CNS of PBS - DTX - treated 2d2 . tdRFPTh17 - transferred CD11c - DTR / GFP → C57BL / 6mice 2 - 3 d after onset of disease . Upper panels : were stimulated with plate - bound anti - CD3 / anti - CD28 , stained for CD4 cytokines IFN - y , IL - 17 ( pre - gated in addition to FSC / SSC on CD45 + CD4 + ) . Lower panels : expression of transcription factor FoxP3 ; pooled data from three , representative for two independent experiments . ( D ) , ( E ) of cytokine expression data as shown in ( C ) for pooled data from 2 independent experiments ; Mann - Whitney - U - * p < 0 . 05 .

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(C) Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFPTh17-transferred CD11c-DTR/GFP→C57BL/6mice 2-3 d after onset of the disease. Upper panels: cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-y, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3; pooled data from three animals, representative for two independent experiments.(D), (E) Quantification of cytokine expression data as shown in (C) for pooled data from 2 independent experiments; Mann-Whitney-U-Test * p < 0.05.


( F ) 2d2Th17 were transferred into lymphopenic Rag2 - / - c - gamma - / - . Mononuclear were isolated from spleen brain at peak of disease analyzed for IL - 17 GM - CSF production after stimulation with anti - CD3 / anti - CD28 . ( G ) GM - CSF production of IL - 17 producers versus IL - 17 non - producers of CNS - isolated 2d2Th17 ( see also ( F ) ) . Each dot / square represents a single ; Kruskal - Wallis - ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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(F) 2d2Th17 were transferred into lymphopenic Rag2-/-c-gamma-/-. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28.(G) GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2Th17 cells (see also (F)). Each dot/square represents a single animal; Kruskal-Wallis-test; * p < 0.05, ** p < 0.01, **** p < 0.0001.


( B ) Microarray signal intensities of strongest regulated genes from ( A ) are shown in detailed statistical for different T subgroups . Values are depicted as signal mean ± from three independent experiments . Statistical significance was determined using one - way ANOVA with post - hoc Tukey for multiple comparisons . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(B) Microarray signal intensities of strongest regulated genes from (A) are shown in detailed statistical analysis for the different T cell subgroups. Values are depicted as signal intensity mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA with post-hoc Tukey Test for multiple comparisons. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


TPLSM of EAE lesions in brainstem of adoptive transfer EAE at onset of disease . EAE was induced by transfer of in vitro differentiated 2d2Th17 ( tdRFP - red , IL - 17 - EGFP - green ) into CD11c - DTR / GFPmice ( CD11c - GFP - green ) ; imaging 300 × 300 µm . ( A ) Intravascular IL - 17hi2d2Th17 ( arrow , double positive - > green : IL - 17EGFP red : 2d2 . tdRFP ) rolling towards a CD11c - GFP + ( arrowhead ) . dotted lines mark a venous vessel . ( B ) Time - lapse TPLSM ( maximal projections ) of boxed in ( A ) shows that perivascular elongated CD11c - GFP + ( asterisk ) enters into contact with IL - 17 - expressing ( GFP - green 2d2 . tdRFP - red double - positive ; arrow arrow head ) Th17 ( scale bar : 20 µm ) . ( C ) XYZ - resolved TPLSM depiction reveals that elongated perivascular CD11c - GFP + makes contact with intravascular 2d2Th17 via a filopodium - like dendrite ( xy - plane , 300 × 300 µm , z - depth 70 µm ) .

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TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2Th17 cells (tdRFP-red, IL-17-EGFP-green) into CD11c-DTR/GFPmice (CD11c-GFP - green); imaging area 300 × 300 µm.(A) Intravascular IL-17hi2d2Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) rolling cell towards a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel.(B) Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (asterisk) enters into contact with IL-17-expressing (GFP - green and 2d2.tdRFP - red double-positive; arrow and arrow head) Th17 cells (scale bar: 20 µm).(C) XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 µm, z-depth 70 µm).


( D ) Strong interaction of a perivascular IL - 17hi2d2Th17 with a perivascular CD11c - GFP + ( insert upper left contact ) . ( E ) Time lapse of insert in ( D ) : Stopping motility of round - shaped IL - 17hi2d2Th17 is followed by entry into CNSparenchyma . ( F ) Magnified image of interaction of upper left ( G ) lower right perivascular T - DC contacts as shown in ( D ) ( E ) .

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(D) Strong interaction of a perivascular IL-17hi2d2Th17 with a perivascular CD11c-GFP+ cell (insert upper left contact).(E) Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi2d2Th17 cell is followed by entry into the CNSparenchyma.(F) Magnified image of interaction of the upper left and (G) lower right perivascular T cell-DC contacts as shown in (D) and (E).


( H ) Time - lapse imaging of parenchymal CD11c - GFP + ( arrowhead ) , targeted migration of an IL - 17hi 2d2 Th17 ( arrow , double positive - > green : IL - 17EGFP red : 2d2 . tdRFP ) towards CD11c - GFP + ( arrowhead ) . Scale bar is 20 µm .

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(H) Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) towards the CD11c-GFP+ cell (arrowhead). Scale bar is 20 µm.


( I ) of motility pattern of IL - 17hi 2d2 . tdRFP vs . 2d2 . tdRFP in CD11c - DTR / GFP brainstem lesions before after ( 2 - 5 h ) intraperitoneal DTX injection . Evaluation of mean track velocity of single tracking ( each dot represents a track ) ; pooled data from at least two independent experiments ( > 3 imaging areas ) . For statistical , Mann - Whitney - U - was performed ( * * * p < 0 . 0001 , n . s . = not significant ) . ( K ) of stopping ( red bar ; instantaneous velocity < 2 µm / min ) of data set as shown in ( I ) .

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(I) Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2-5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single cell tracking (each dot represents a track); pooled data from at least two independent experiments (>3 imaging areas). For the statistical analysis, the Mann-Whitney-U-test was performed (*** p < 0.0001, n.s. = not significant).(K) Percentage of stopping cells (red bar; instantaneous velocity <2 µm/min) of the data set as shown in (I).


Active EAE was induced by subcutaneous immunization with CFA / MOG35 - 55 . were isolated from CNS of EAE at different stages of disease ( pre - onset [ day 8 - 9 ] , peak [ day 14 - 17 ] , chronic [ day 22 - 27 ] ; data from at least two independent experiments ) . Representative samples are shown , which were analyzed by flow cytometry for expression of markers CD11c , CD11b Ly6C / G ( pre - gated on lympho - monocytic by FSC / SSC ; IAb + expression was confirmed for CD11c + in separate stainings ) . ( A ) In CNS , there was a strong increase of CD11c + proportions over different phases of disease . composition of CD11c + subpopulations changed over time , starting with a preponderance of CD11c + Ly6C / G - conventional DCs before onset of EAE . In peak chronic phase there was a continuous increase in proportion of monocyte - derived CD11c + . ( B ) For CNS , of absolute numbers shows a strong increase in CD11c + during course of EAE . ( C ) In CNS , proportions of subpopulations show an increasing proportion of monocyte - derived CD11c + .

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Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8-9], peak [day 14-17], chronic [day 22-27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.


( A ) CD11c + , CD4 + CX3CR1 + CD45intIAb + ( microglia ) were isolated from peak EAE from CNS . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleen CD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . CNS CD11c + show gene expression of inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Il23 ( mean ± ) . ( C ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in CNS samples as described

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(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleen CD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNS CD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Il23 (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above


( B ) CD11c + , CD4 + CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . Relevantly regulated chemokines in other populations than CNS CD11c + are shown ( mean ± ) . ( D ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in spleen samples .

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(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). Relevantly regulated chemokines in other cell populations than CNS CD11c+ cells are shown (mean ± SEM).(D) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the spleen samples.


Figure 5Chemokine expression profiling of CD11c + in spleen CNS reveal a key role of CNSCD11c + for inflammatory chemokines . ( A ) CD11c + , CD4 + CX3CR1 + CD45intIAb + ( microglia ) were isolated from peak EAE from CNS . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . CNSCD11c + show gene expression of inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Ccl20 ( mean ± ) . ( B ) CD11c + , CD4 + CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Relevantly regulated chemokines in other populations than CNSCD11c + are shown ( mean ± ) . ( C ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in CNS samples as described above ( D ) spleen samples . Statistical significance was determined using Kruskal - Wallis . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Figure 5Chemokine expression profiling of CD11c+ cells in the spleen and CNS reveal a key role of CNSCD11c+ cells for inflammatory chemokines.(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNSCD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Ccl20 (mean ± SEM).(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Relevantly regulated chemokines in other cell populations than CNSCD11c+ cells are shown (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and (D) the spleen samples.Statistical significance was determined using Kruskal-Wallis test. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


( A ) CD11c - GFP + DC cluster formation near venous vessel with ( B ) protrusions attracting 2d2 . tdRFP Th17 .

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(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells.


( C ) Low - velocity motility of 2d2 . tdRFPT in DC cluster compared to outside of cluster . SHG in upper panel on left ( arrow ) . In addition , intercellular matrix ( arrow ) are visualized that are only found in inflamed CNSparenchyma are due to second harmonic generation ( SHG ) at 1110 nm wavelength excitation detection at 535 ± 50nm . Tracking of 2d2 . tdRFPTh17 in right panel ( color - coded tracks , low velocity : red , high velocity : yellow ) . ( D ) Quantitative of T tracking : higher mean velocity of 2d2 . tdRFPTh17 in onset of disease with differential distribution at peak of disease . Cluster associated tracks have significantly lower mean velocities than Th17 outside CD11c - GFP clusters . ( E ) Analogously for track straightness ; Kruskal - Wallis Dunn ' s Multiple Comparison ( * * * p < 0 . 0001 ) were performed ; pooled data from at least three independent experiments per group .

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(C) Low-velocity motility of 2d2.tdRFPT cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFPTh17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn's Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.


A . unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15 - 18 h post - injection . arrowheads represent polar ( PB ) . PN represents pronucleus .

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A. The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15-18 h post-injection. The white arrowheads represent the polar body (PB). PN represents pronucleus.


B . expression localization of PLCζ in sperm from affected individuals .

B. The expression and localization of PLCζ in sperm from the affected individuals.


C . H & E of spermatozoa from affected individuals . black arrow represents normal spermatozoa . black arrowheads represent tapered - head spermatozoa with increased major axis normal minor axis . arrowheads represent thin spermatozoa with decreased major axis minor axis .

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C. H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length.


D . ultrastructure of spermatozoa from normal affected . squares represent perinuclear ring . Ac represents acrosome . N represents nucleus .

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D. The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus.


E . range of acrosomes in sperm heads from normal affected ( n = 7 ) .

E. The range of acrosomes in sperm heads from normal and affected individual (n=7).


A . Fertility assessment experiments in WT Iqcn - / - mice after mating with WT mice ( n = 18 ) .

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A. Fertility assessment experiments in WT and Iqcn-/- male mice after mating with WT female mice (n = 18).


B . of testes epididymides in WT Iqcn - / - mice .

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B. Size of the testes and epididymides in WT and Iqcn-/- male mice.


C . of testis / in WT Iqcn - / - mice ( n = 3 ) .

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C. Quantification ratio of the testis weight/body weight in WT and Iqcn-/- male mice (n = 3).


D . Sperm in caudal epididymis from WT Iqcn - / - mice ( n = 3 ) .

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D. Sperm counts in the caudal epididymis from WT and Iqcn-/- male mice (n = 3).


E . H & E of sperm from WT Iqcn - / - mice . Red arrowheads represent sperm with no sickle shape heads .

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E. H&E staining of sperm from WT and Iqcn-/- male mice. Red arrowheads represent sperm with no sickle shape heads.


F . of sperm without sickle shape heads from WT Iqcn - / - mice ( n = 3 ) .

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F. Percentage of sperm without sickle shape heads from WT and Iqcn-/- male mice (n = 3).


G . Ultrastructure of spermatozoa from WT Iqcn - / - mice . Ac represents acrosome .

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G. Ultrastructure of spermatozoa from WT and Iqcn-/- male mice. Ac represents the acrosome.


H . Immunostaining of lectin ( green ) in spermatozoa from WT Iqcn - / - mice .

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H. Immunostaining of lectin (green) in spermatozoa from WT and Iqcn-/- male mice.


I . of abnormal acrosomes in spermatozoa from WT Iqcn - / - mice ( n = 3 ) .

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I. Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn-/- male mice (n = 3).


J . of spontaneous induced acrosome reactions in spermatozoa from WT Iqcn - / - mice ( n = 3 ) .

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J. Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn-/- male mice (n = 3).


A . Representative two pronucleus ( 2PN ) zygotes , two - , blastocysts after ICSI in WT Iqcn - / - mice .

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A. Representative two pronucleus (2PN) zygotes, two-cell embryos, and blastocysts after ICSI in WT and Iqcn-/- male mice.


B . of 2PN zygotes , two - blastocysts after ICSI in WT Iqcn - / - mice ( n = 3 ) .

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B. Percentage of 2PN zygotes, two-cell embryos and blastocysts after ICSI in WT and Iqcn-/- male mice (n = 3).


C . Profile of Ca2 + responses induced by oocyte injection with spermatozoa from WT Iqcn - / - mice ( n = 5 ) . Spermatozoa from WT mice induced 8 - 9 Ca2 + spikes over a 3 - h period ( blue ) , while spermatozoa from Iqcn - / - mice failed to induce spikes ( red ) .

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C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).


D . Immunostaining of oocytes injected with spermatozoa from WT Iqcn - / - mice . expression of PLCζ ( red ) α - tubulin ( green ) was detected in oocytes . M represents nucleus F represents nucleus .

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D. Immunostaining of oocytes injected with spermatozoa from WT and Iqcn-/- male mice. The expression of PLCζ (red) and α-tubulin (green) was detected in oocytes. M represents male nucleus and F represents the female nucleus.


E . Expression localization of PLCζ in sperm from WT Iqcn - / - mice .

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E. Expression and localization of PLCζ in sperm from WT and Iqcn-/- male mice.


F . Expression localization of PLCζ during acrosomal biogenesis from WT Iqcn - / - mice . PLCζ was localized in acroplaxome in WT spermatids . PLCζ was only localized in tip of head in cap acrosome phases ( arrowheads ) was lost during maturation phase in Iqcn - / - mice .

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F. Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn-/- male mice. PLCζ was localized in the acroplaxome in WT spermatids. PLCζ was only localized in the tip of the head in the cap and acrosome phases (white arrowheads) and was lost during the maturation phase in Iqcn-/- mice.


A . Expression localization of IQCN during spermiogenesis in testes of WT mice . IQCN ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 . Sb represents late spermatids . Scale bars , 20 μm .

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A. Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with α-tubulin (green) from steps 8-14. Sb represents late spermatids. Scale bars, 20 μm.


B . Ultrastructure of testes from WT Iqcn - / - mice from steps 8 - 16 . MA represents manchette . PR represents perinuclear ring . CD represents droplets . Scale bars , 2 μm ( steps 8 - 12 ; 15 - 16 ) 5 μm ( steps 13 - 14 ) .

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B. Ultrastructure of the testes from WT and Iqcn-/- male mice from steps 8-16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 μm (steps 8-12; 15-16) and 5 μm (steps 13-14).


C . Immunostaining of α - tubulin ( green ) in spermatids at steps 13 - 14 . MA represents manchette . PR represents perinuclear ring .

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C. Immunostaining of α-tubulin (green) in spermatids at steps 13-14. MA represents manchette. PR represents perinuclear ring.


D . of manchette structure in spermatids at steps 13 - 14 from WT Iqcn - / - mice ( n = 10 ) .

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D. Length of the manchette structure in spermatids at steps 13-14 from WT and Iqcn-/- male mice (n = 10).


A . Expression localization of calmodulin during spermiogenesis in testes of WT Iqcn - / - mice . Calmodulin ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 in WT mice , but failed to co - localize in steps 13 - 14 in Iqcn - / - mice .

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A. Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn-/- male mice. Calmodulin (red) was co-localized with α-tubulin (green) from steps 8-14 in WT mice, but failed to co-localize in steps 13-14 in Iqcn-/- male mice.


B . Interaction between IQCN calmodulin ( CaM ) following co - IP by IQCN antibody in testes from WT Iqcn - / - mice ( n = 3 ) . IQCN interacts with calmodulin in WT mice , interaction of which is completely lost in Iqcn - / - mice . GAPDH was used as internal control .

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B. Interaction between IQCN and calmodulin (CaM) following co-IP by IQCN antibody in testes from WT and Iqcn-/- male mice (n = 3). IQCN interacts with calmodulin in WT mice, the interaction of which is completely lost in Iqcn-/- male mice. GAPDH was used as the internal control.


C . Immunostaining of calmodulin ( red ) α - tubulin ( green ) in HeLa . normal ( WT ) mutant ( c . 910C > T c . 2453 _ 2454del ) plasmids of IQCN were transfected into HeLa . HeLa were treated with an antagonist of calmodulin W - 7 . Multipolar spindles were observed in HeLa transfected with mutant plasmids W - 7 treatment .

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C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment.


D . of multipolar spindles in WT mutant IQCN transfected HeLa . ( n = 3 biological replicates )

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D. Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells. (n = 3 biological replicates)


C ) , of pups gathered in nest location is significantly reduced in Shank2 - / - mice . Mann - Whitney , * * * P < 0 . 001 . Data information : data are presented as mean ± .

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C), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM.


( H ) Shank2 + / + Shank2 - / - mice displayed a significant preference for novel object versus familiar one in session , two - way mixed ANOVA , effect of object : * * * P < 0 . 001 , effect of genotype : P = 0 . 167 , object x genotype interaction : P = 0 . 667 , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : graphs are presented as mean ± , NS : not significant .

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(H) Shank2+/+ and Shank2-/- mice displayed a significant preference for the novel object versus the familiar one in the test session, two-way mixed ANOVA, effect of object: ***P<0.001, effect of genotype: P=0.167, object x genotype interaction: P=0.667, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All graphs are presented as mean ± SEM, NS: not significant.


( J ) Left panel : Electron of posterior pituitary from Shank2 + / + Shank2 - / - mice in lower ( left panel ) higher magnification . Scale bar ( 500 nm ) . Right diagram : No significant difference was detected in number of vesicles in posterior pituitary between Shank2 + / + Shank2 - / - mice , Mann - Whitney , P = 0 . 507 , Shank2 + / + n = 3 , Shank2 - / - n = 3 . ( K - M ) Additionally , Shank2 - / - mice display no significant difference in : ( K ) Estradiol - , Mann - Whitney , P = 0 . 475 , Shank2 + / + n = 6 , Shank2 - / - n = 7 , ( L ) Progesterone , unpaired , two - Student ' s t - , P = 0 . 570 , Shank2 + / + n = 5 , Shank2 - / - n = 5 or ( M ) Prolactin - plasma concentrations , unpaired , two - Student ' s t - , P = 0 . 980 ; Shank2 + / + n = 3 , Shank2 - / - n = 3 . Data information : data are presented as mean ± , NS : not significant . E , endothelium ; CL , capillary lumen ; M , mitochondria ; NG , neurosecretory granule ; AE , axonal endings , HYP , Hypothalamus ; PIT , Pituitary .

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(J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.


( A - G ) qRT - PCR of mRNA expression of hormonal genes hormonal receptors normalized to house - keeping gene HMBS of 9 - 10 week old naive postnatal day 1 Shank2 + / + Shank2 - / - mice : ( A ) Oxytocin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 335 . ( B ) Vasopressin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 945 . ( C ) Prolactin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 202 . Additionally , no significant difference was evident in ( D ) oxytocin receptor , one - way ANOVA , P = 0 . 675 , ( E ) prolactin receptor , one - way ANOVA , P = 0 . 912 , ( F ) vasopressin receptor , one - way ANOVA , P = 0 . 418 ( G ) estrogen receptor alpha expression , one - way ANOVA , P = 0 . 890 in comparison to Shank2 + / + mice . data are presented as mean ± , NS : not significant . Pituitary , ( n = 3 pituitary pooled per value , n = 3 hypothalamus ( Oxytocin , Vasopressin , Oxytocin receptor , Vasopressin receptor , estrogen alpha ) n = 2 - 3 hypothalamus ( Prolactin receptor ) ) . HYP , hypothalamus ; PIT , pituitary .

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(A-G) qRT-PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house-keeping gene HMBS of 9-10 week old naive and postnatal day 1 Shank2+/+ and Shank2-/- mice: (A) Oxytocin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.335. (B) Vasopressin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.945. (C) Prolactin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.202. Additionally, no significant difference was evident in (D) oxytocin receptor, one-way ANOVA, P=0.675, (E) prolactin receptor, one-way ANOVA, P=0.912, (F) vasopressin receptor, one-way ANOVA, P=0.418 and (G) estrogen receptor alpha expression, one-way ANOVA, P=0.890 in comparison to Shank2+/+ mice. All data are presented as mean ± SEM, NS: not significant. Pituitary gland, (n=3 pituitary glands pooled per value, n=3 hypothalamus (Oxytocin, Vasopressin, Oxytocin receptor, Vasopressin receptor, estrogen alpha) n=2-3 hypothalamus (Prolactin receptor)). HYP, hypothalamus; PIT, pituitary gland.


( E - H ) Repeated pup exposure does not induce major components of social attachment behavior in Shank2 - / - : ( E ) pup grooming , unpaired , two - Student ' s t - , * * * P < 0 . 001 ( F ) crouching , Mann - Whitney , * * * P < 0 . 001 ( G ) nest building , Mann - Whitney , * * * P < 0 . 001 , ( H ) maternal interaction , unpaired , two - Student ' s t - , * * * P < 0 . 001 ( trial 5 ) , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : data are presented as mean ± , NS : not significant .

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(E-H) Repeated pup exposure does not induce major components of social attachment behavior in Shank2-/- females: (E) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (F) crouching, Mann-Whitney test, ***P<0.001 and (G) nest building, Mann-Whitney test, ***P<0.001, (H) maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001 (trial 5), Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant.


( L ) of synaptic in crude synaptosomes fraction ( P2 ) from hypothalamus of Shank2 + / + Shank2 - / - mice . Shank2 + / + n = 5 ( black bars ) , Shank2 - / - n = 5 ( blue bars ) . Unpaired , two - Student ' s t - .

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(L) Levels of synaptic proteins in the crude synaptosomes fraction (P2) from the hypothalamus of Shank2+/+ and Shank2-/- mice. Shank2+/+ n = 5 (black bars), Shank2-/- n = 5 (blue bars). Unpaired, two-tailed Student's t-test.