D . Miro shRNAs mitigate the effect of A & O on EYFP - Parkin translocation . PC6 cells transfected with EYFP - Parkin with and without Miro shRNAs were treated with DMSO or A & O ( both 10 µM ) for 3h . The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs - expressing group when compared with the scrambled shRNA - expressing group . * * * * P < 0 . 0001 , n = 4 dishes , 20 fields per dish , one - way

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D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA


E . Miro shRNAs inhibit PINK1 - induced mitophagy in PC6 cells . The figure shows the changes in the excitation spectra of mitochondrially targeted pH - sensitive Keima ( ratio of 561 nm / 458nm fluorescence intensities ) . * * * * P < 0 . 0001 , n = 80 cells from 4 dishes , Kruskal - Wallis

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E. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis test


F . Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima . Mitochondria in lysosomes ( acidic pH ) were counted manually , by a blinded observer ( * * P < 0 . 01 , n = 9 dishes , 9 fields per dish , from 2 independent experiments , one - way ANOVA )

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F. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA)


G . Miro shRNAs decrease the number of mitochondria co - localised with the autophagosome marker EGFP - LC3B in primary cortical neurons ( * * P < 0 . 01 , n = 5 dishes , 9 fields per dish , one - way ANOVA ) .

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G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).


A . The pattern of Parkin translocation caused by Miro1 overexpression is different from A & O - induced translocation . The figure illustrates puncta - like EYFP - Parkin on mitochondria in antimycin and oligomycin - treated ( both 10 µM for 3h ) PC6 cells ( middle panels ) , whereas in Miro1 - overexpressing cells , EYFP - Parkin appears along rod - shaped mitochondria visualised with CFP - mito ( lower panels ) . The merged panels present EYFP - Parkin ( green ) and CFP - mito ( red )

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A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 µM for 3h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red)


B . Overexpression of myc - Miro1 and untagged Miro2 , but not myc - Miro2 , induce EYFP - Parkin translocation to mitochondria . * * * * P < 0 . 0001 vs control group , n = 5 - 6 dishes , 20 fields per dish , one - way ANOVA or t -

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B. Overexpression of myc-Miro1 and untagged Miro2, but not myc-Miro2, induce EYFP-Parkin translocation to mitochondria. ****P < 0.0001 vs control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA or t-test


C . Quantification of EYFP - Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria , which is significantly weaker than PINK1 overexpression - or A & O ( 3 h ) - induced translocation . * * P < 0 . 01 and * * * * P < 0 . 0001 vs control group , n = 18 - 32 cells from 3 dishes per group , Kruskal - Wallis

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C. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria, which is significantly weaker than PINK1 overexpression- or A&O (3 h)-induced translocation. **P < 0.01 and ****P < 0.0001 vs control group, n = 18-32 cells from 3 dishes per group, Kruskal-Wallis test


Miro1 overexpression does not cause mitochondrial depolarization ( D ) Representative image of Miro1 and EYFP - Parkin - transfected cells stained with TMRE . The merged panels present EYFP - Parkin ( green ) and TMRE ( red )

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Miro1 overexpression does not cause mitochondrial depolarization (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red)


Miro1 overexpression does not cause mitochondrial depolarization ( E ) Relative TMRE intensity quantified in neurons transfected with Miro1 ( detected by CFP - mito co - transfection ) ; the control group is non - transfected cells in the same image . The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs - expressing neurons . Note that FCCP treatment ( 3 μM for 15 min ) almost completely abolished TMRE fluorescence . * * * * P < 0 . 0001 vs control group , n = 94 - 186 cells from 4 dishes per group , Kruskal - Wallis

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Miro1 overexpression does not cause mitochondrial depolarization (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 vs control group, n = 94-186 cells from 4 dishes per group, Kruskal-Wallis test


F . Co - expression of HA - Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons . Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity . Co - transfection of Parkin did not affect significantly the motility parameters . Data are presented as Tukey boxplot . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 , n = 689 - 846 individual mitochondria from 39 - 44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments , Kruskal - Wallis

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F. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689-846 individual mitochondria from 39-44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal-Wallis test


G . Parkin overexpression does not induce degradation of myc - Miro1 in control conditions . Representative Western blot image and analysis of myc - Miro1 expression in HEK cells . Treatment with A & O ( both 15 µM ) for 3h in the absence of MG132 led to slight degradation of myc - Miro1 . Co - expression of EYFP - Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present . However , Parkin co - expression enhanced Miro1 degradation when cells were treated with A & O . * P < 0 . 05 and * * * * P < 0 . 0001 , ns : not significant , n = 4 independent experiments , one way ANOVA . Note that only the main band of myc - Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP -

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G. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 µM) for 3h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-Parkin


H . EYFP - Parkin co - immunoprecipitates with overexpressed myc - Miro1 , both in DMSO - or A & O ( both 15 µM for 3h ) treated HEK

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H. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- or A&O (both 15 µM for 3h) treated HEK cells


I . Endogenous Parkin co - immunoprecipitates with endogenous Miro1 . Miro1 was immunoprecipitated from non - transfected and non - treated HEK cells using mouse monoclonal anti - Miro1 antibody and immunoblotted for endogenous Parkin .

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I. Endogenous Parkin co-immunoprecipitates with endogenous Miro1. Miro1 was immunoprecipitated from non-transfected and non-treated HEK cells using mouse monoclonal anti-Miro1 antibody and immunoblotted for endogenous Parkin.


A . Miro1 induces EYFP - Parkin translocation in the presence of PINK1 shRNA . PC6 cells were transfected with EYFP - Parkin and Miro1 or treated for 3h with A & O ( both 10 µM ) in the presence of scrambled shRNA or PINK1 shRNA . Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A & O treatment did not . * * * * P < 0 . 0001 compared with the respective control group , n = 5 - 6 dishes , 20 fields per dish , one - way

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A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA


B . Miro1 induces EYFP - Parkin translocation in PINK1 - / - MEFs . MEFs obtained from PINK1 + / + and - / - mice were transfected with EYFP - Parkin and Miro1 or treated with A & O ( both 10 µM for 3h ) . Miro1 induced Parkin translocation in both MEF lines but A & O only in PINK1 + / + MEFs . * * * * P < 0 . 0001 compared with respective control group , n = 6 dishes , one - way

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B. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVA


C . Mutated EYFP - Parkin is recruited to mitochondria in response to Miro1 overexpression . PC6 cells were transfected with wild type , T240R or C431N EYFP - Parkin and Miro1 or treated for 3 hours with A & O ( both 10 µM ) . Miro1 induced translocation of all EYFP - Parkin mutants whereas A & O treatment was not able to induce equivalent translocation of Parkin mutants . * * P < 0 . 01 and * * * * P < 0 . 0001 compared with respective control group , n = 3 - 6 dishes , 20 fields per dish , one - way

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C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA


D . Parkin mutants T240R and C431N interact with Miro1 both in DMSO - or A & O - treated ( both 15 µM for 3h ) HEK cells . Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin - expressing groups .

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D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.


A . Miro1 K572R mutant interacts with HA - Parkin similarly to Miro1 WT . HEK cells overexpressing myc - Miro1 WT or K572R mutant were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated and probed for myc - Miro1 and HA - Parkin . Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP , which is missing in the case of the K572R

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A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant


B . Miro1 K572R ubiquitination by Parkin is reduced . HEK cells overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 K572R and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin - HA and myc -

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B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1


C . Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP - Parkin . K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A & O ( both 7 . 5 µM ) for 3 hours . Note the spatial heterogeneity values shown in the

C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the images


D . Quantification of EYFP - Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A & O ( both 7 . 5 µM ) treatment for 3 hours , but K572R mutation of Miro1 abolishes this effect . * * * P < 0 . 001 and * * * * P < 0 . 0001 , n = 152 - 172 cells from 3 dishes per group , Kruskal - Wallis test . Data are presented as means ± SEM .

D. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.


A . A Ca2 + - insensitive mutant of Miro1 ( Miro1 - EF ) interacts with Parkin . HEK cells overexpressing myc - Miro1 or myc - Miro1 - EF and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 ( red ) and Parkin ( green ) . The high - contrast grayscale image from the green channel of this blot shows that endogenous Parkin co - immunoprecipitated with Miro1 in all conditions . The bar chart shows the quantification of co - immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP - Parkin signal ( * P < 0 . 05 and * * P < 0 . 01 , repeated measures

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A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVA


B . Miro1 - EF is less susceptible to ubiquitination by Parkin . HEK cells overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 - EF and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin ( red ) , Miro1 ( green ) and Parkin ( green ; representative images are from the same experimental material on separate SDS - PAGE - WB membranes ) . The high - contrast grayscale shows the green channel of this blot , and the bar chart shows quantification of co - immunoprecipitated Miro from 3 different blots ( * * P < 0 . 01 and * * * P < 0 . 001 , repeated measures ANOVA ) . Note that EYFP - Parkin co - immunoprecipitated with ubiquitin only after A & O

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B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment


C . Mutation of Miro1 EF - hands diminishes Miro1 ubiquitination with S65 - phosphorylated ubiquitin . HEK cells were transfected with EYFP - Parkin with either myc - Miro1 - WT or myc - Miro1 - EF with or without PINK1 - V5 . Cells were treated with either DMSO or A & O ( both 10 µM ) for 5 hrs with the exception of the PINK1 - V5 group . Myc - Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 ( red ) , S65 - Phosphoubiquitin and PINK1 - V5 ( green ; the weak bands marked with the asterisk are nonspecific ) .

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C. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).


A . Kinetics of EYFP - Parkin translocation in response to A & O is different in control and Miro1 - EF - expressing PC6 cells . The inset shows how different compartments of the same dish were transfected with EYFP - Parkin and control plasmid or Miro1 - EF and followed over 3 hrs using a confocal microscope after addition of A & O ( both 10 µM ) . The graph shows the average spatial heterogeneity of EYFP - Parkin fluorescence , reflecting the ratio of mitochondrial - to - cytosolic Parkin over time ( n = 40 - 43 cells , difference between the curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM )

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A. Kinetics of EYFP-Parkin translocation in response to A&O is different in control and Miro1-EF-expressing PC6 cells. The inset shows how different compartments of the same dish were transfected with EYFP-Parkin and control plasmid or Miro1-EF and followed over 3 hrs using a confocal microscope after addition of A&O (both 10 µM). The graph shows the average spatial heterogeneity of EYFP-Parkin fluorescence, reflecting the ratio of mitochondrial-to-cytosolic Parkin over time (n = 40-43 cells, difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


The percentage of cells where EYFP - Parkin translocates in response to A & O ( both 10 µM , B is decreased in Miro1 - EF - expressing PC6 cells . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with the indicated group ( n = 3 - 6 dishes , 20 fields per dish , one way ANOVA

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The percentage of cells where EYFP-Parkin translocates in response to A&O (both 10 µM, B is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dish, one way ANOVA for


The percentage of cells where EYFP - Parkin translocates in response t PINK1 overexpression ( C ) is decreased in Miro1 - EF - expressing PC6 cells . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with the indicated group ( n = 3 - 6 dishes , 20 fields per dis Welch " s ANOVA

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The percentage of cells where EYFP-Parkin translocates in response t PINK1 overexpression (C) is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dis Welch"s ANOVA f


D . Time course showing laser - induced EYFP - Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons . Cells were transfected with CFP - mito and EYFP - Parkin and visualized using a confocal microscope . Mitochondria were irradiated in a 0 . 9 µm x 0 . 9 µm - frame using a 405 - nm laser line ( 25 iterations using 100 % laser power , pixel time 164 µs )

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D. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs)


E , F Miro1 - EF delays and reduces irradiation - induced EYFP - Parkin translocation . ( E ) The time course of translocation strength in control and Miro1 - EF - expressing neurons . Difference between the curves P = 0 . 0016 , two - way ANOVA . ( F ) Comparison at the 60 - min time point ( * P < 0 . 05 , n = 6 dishes , at least 9 neurons per dish , t - test ) .

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E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).


A . EYFP - Parkin translocation requires Ca2 + . PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2 + or in Ca2 + free Krebs containing 5 μM BAPTA - AM , were treated with A & O ( both 10 μM ) . Images ( left panels ) demonstrate that EYFP - Parkin translocation was significantly weaker at zero Ca2 + at 195 min after A & O treatment ( compared with 15 min ; note the spatial heterogeneity values shown in photos ) . Spatial heterogeneity analysis ( right panel ) demonstrated that Parkin translocation was significantly decelerated in zero Ca2 + conditions ( n = 25 - 27 cells ; difference between the curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM )

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

A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


B . EYFP - Parkin translocation accelerates in the presence of the L - type Ca2 + channel agonist Bay K 8644 ( 5 μM ; n = 19 - 21 cells ; difference between the curves p < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM )

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B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into the cytosol , visualised by increased cytosolic GCaMP7b

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into the cytosol Glutamate - induced Ca2 + entry was not affected by Miro1 WT or Miro1 EF overexpression ; n = 31 - 48 neurons per group ; ns , Kruskal - Wallis test )

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)


E , F Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) led to the appearance of a few Parkin - positive strong puncta co - localising with mitochondrial marker ( white arrow ) , imaged 2 . 5 h after treatment ( E ) . Further analysis demonstrated that the expression of Miro1 - EF mutant decreased and Miro1 WT increased the number of neurons with EYFP - Parkin - positive mitochondria following glutamate treatment ( F ; n = 6 - 10 ; * * P < 0 . 01 and * * * * P < 0 . 0001 , one - way ANOVA )

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E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)


G . Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) was associated with an increased number of mitochondrial co - localisation with the autophagosome marker EGFP - LC3B in neuronal soma at 5 - 6 h later , which was not observed in Miro1 - EF - expressing neurons ( n = 8 dishes ; * P < 0 . 05 , one - way ANOVA )

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G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)


H . Glutamate treatment ( 30 μM glutamate and 1 μM glycine for 10 min ) was also associated with a significant loss of mitochondrial density in axons at 6 h later , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 60 - 90 axons from 6 - 9 dishes ; * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 , Kruskal - Wallis test )

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H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)


I . Glutamate treatment ( 100 μM glutamate and 10 μM glycine for 10 min ) led to a decline , at 6 - 8 h , in cytosolic ATP measured using ATP / ADP sensor Perceval HR , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 59 - 60 neurons from 6 dishes ; * * P < 0 . 01 , Welch " s

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

I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s A


J . Glutamate ( 100 μM glutamate and 10 μM glycine for 10 min ) induced excitotoxicity ( 24 h after treatment ) that was partially reversed by Miro1 - EF but not by Miro1 WT . Data is presented as a percentage of control neuron count ( n = 10 dishes ; * * * P < 0 . 001 , repeated measures ANOVA ) .

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J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).


HSC ( 100 cells ) were untreated or cultured under pH 7 . 4 and pH 6 . 9 for 40 h and transplanted with competitor cells . n = at least 18 mice per group out of at least 7 experiments ( biological repeats ) Representative FACS dot plots and quantitative and statistical analysis of LT - HSC , ST - HSC , and LMPP distribution among donor derived LSKs in primary ( I ) recipients . * p < 0 . 05 using two - tailed unpaired Student ' s t - test .

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HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs in primary (I) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.


HSC ( 100 cells ) were untreated or cultured under pH 7 . 4 and pH 6 . 9 for 40 h and transplanted with competitor cells . n = at least 18 mice per group out of at least 7 experiments ( biological repeats ) Representative FACS dot plots and quantitative and statistical analysis of LT - HSC , ST - HSC , and LMPP distribution among donor derived LSKs secondary ( J ) recipients . * p < 0 . 05 using two - tailed unpaired Student ' s t - test .

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HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs secondary (J) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.


( C ) Representative flow cytometric analyses of EdU incorporation and DNA content in HSCs after culture under pH 7 . 4 and pH 6 . 9 conditions for 40 h . Percentage of cells in G1 , S and G2 / M phase of cell cycle under pH 7 . 4 or pH 6 . 9 conditions . ( n = 4 biological replicates , * * * p < 0 . 001 ; using two - tailed unpaired Student ' s t - test )

(C) Representative flow cytometric analyses of EdU incorporation and DNA content in HSCs after culture under pH 7.4 and pH 6.9 conditions for 40 h. Percentage of cells in G1, S and G2/M phase of cell cycle under pH 7.4 or pH 6.9 conditions. (n=4 biological replicates, ***p < 0.001; using two-tailed unpaired Student's t-test)


( E ) Relative levels of expression of the Hif1α transcriptional targets Pdk - 1 and Vegfa ( n = 4 biological replicates , * p < 0 . 05 ; using two - tailed unpaired Student ' s t - test ) .

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(E) Relative levels of expression of the Hif1α transcriptional targets Pdk-1 and Vegfa (n=4 biological replicates, *p < 0.05; using two-tailed unpaired Student's t-test).


( F ) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7 . 4 or pH 6 . 9 conditions ( Scale bar = 5μm )

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(F) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7.4 or pH 6.9 conditions (Scale bar = 5μm)


( H ) Representative ultra - structural transmission electron microscopy images of untreated and cultured HSCs under pH 7 . 4 or pH 6 . 9 conditions . Data represent n = 20 cells from 2 biological replicates . Arrows label mitochondria , while arrowheads label vacuolar structures ( Scale bar = 1μm ) .

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(H) Representative ultra-structural transmission electron microscopy images of untreated and cultured HSCs under pH 7.4 or pH 6.9 conditions. Data represent n=20 cells from 2 biological replicates. Arrows label mitochondria, while arrowheads label vacuolar structures (Scale bar = 1μm).


( B ) Hierarchical clustering and heatmap representing global visualization of all the DEGs in HSCs present at pH 6 . 9 or pH 7 . 4 for 40 h ( n = 6 biological replicates ) . Results were visualized using Treeview .

(B) Hierarchical clustering and heatmap representing global visualization of all the DEGs in HSCs present at pH 6.9 or pH 7.4 for 40 h (n=6 biological replicates). Results were visualized using Treeview.


( C ) Relative levels of spermidine and spermine in pH 6 . 9 and pH 7 . 4 conditions with respect to untreated samples . n = 3 biological replicates . * p < 0 . 05 and exact p values are mentioned between groups .

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(C) Relative levels of spermidine and spermine in pH 6.9 and pH 7.4 conditions with respect to untreated samples. n=3 biological replicates. *p < 0.05 and exact p values are mentioned between groups.


( E ) Effect of DFMO treatment on pHi of HSCs . , n = 5 biological replicates . * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test .

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(E) Effect of DFMO treatment on pHi of HSCs., n= 5 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.


( G ) Pictures of HSCs after culture at pH 7 . 4 or pH 6 . 9 and in presence or absence of DFMO and SPN for 40 h . Data are representative of 6 biological replicates . Scale bar = 100μm ( H ) Cell volume of HSCs cultured at pH 7 . 4 in the presence of DFMO or in combination with SPN . Data are from > 80 cells per group , representative of 3 biological replicates . * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test .

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(G) Pictures of HSCs after culture at pH 7.4 or pH 6.9 and in presence or absence of DFMO and SPN for 40 h. Data are representative of 6 biological replicates. Scale bar = 100μm (H) Cell volume of HSCs cultured at pH 7.4 in the presence of DFMO or in combination with SPN. Data are from >80 cells per group, representative of 3 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.


( I , J ) Reconstitution level of donor derived - Ly5 . 2 + cells , - lineage distribution in peripheral blood and BM in primary ( I ) and secondary ( J ) recipients from 100 HSCs cultured at pH 7 . 4 in presence of DFMO or in combination of SPN . Data in figure I , J are from n = 9 - 12 recipient mice from 2 independent biological transplantation experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test .

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(I,J) Reconstitution level of donor derived -Ly5.2+ cells, -lineage distribution in peripheral blood and BM in primary (I) and secondary (J) recipients from 100 HSCs cultured at pH 7.4 in presence of DFMO or in combination of SPN. Data in figure I,J are from n= 9-12 recipient mice from 2 independent biological transplantation experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed unpaired Student's t-test.


( K ) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6 . 9 , pH 7 . 4 or pH 7 . 4 in presence of DFMO ( Scale bar = 5μm ) . Bar graphs represent quantification of LAMP2 levels in cells . Data are from > 50 cell representing 3 biological replicates . * * * p < 0 . 001 , * * * * p < 0 . 0001 using two - tailed unpaired Student ' s t - test .

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(K) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO (Scale bar = 5μm). Bar graphs represent quantification of LAMP2 levels in cells. Data are from > 50 cell representing 3 biological replicates. ***p < 0.001, ****p < 0.0001 using two-tailed unpaired Student's t-test.


( L ) Reconstitution level of donor derived Ly5 . 2 + cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6 . 9 , pH 7 . 4 or pH 7 . 4 in presence of DFMO . n = 7 - 9 mice from 2 biological transplantation experiments for murine HSC data . For human HSPC transplants , n = 6 recipient per condition from 3 biological transplantation experiments . * p < 0 . 05 , or exact p value mentioned between groups . Statistic significance was calculated using two - tailed unpaired Student ' s t - test .

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(L) Reconstitution level of donor derived Ly5.2+ cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO. n= 7-9 mice from 2 biological transplantation experiments for murine HSC data. For human HSPC transplants, n= 6 recipient per condition from 3 biological transplantation experiments. *p < 0.05, or exact p value mentioned between groups. Statistic significance was calculated using two-tailed unpaired Student's t-test.


( C ) Period length decreases with continual differentiation of PSCs into HIOs . Maturation of HIOs into kcHIEs leads to 24 - hour rhythms with greater reproducibility . ( D ) The amplitude of oscillations are significantly higher in kcHIEs / bHIEs compared to HIOs . Bmal1 - luc traces in B were generated by plotting the standard deviation of n ≥ 3 biological replicates .

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(C) Period length decreases with continual differentiation of PSCs into HIOs. Maturation of HIOs into kcHIEs leads to 24-hour rhythms with greater reproducibility. (D) The amplitude of oscillations are significantly higher in kcHIEs/bHIEs compared to HIOs. Bmal1-luc traces in B were generated by plotting the standard deviation of n≥3 biological replicates.


( A - C ) mRNA expression of Bmal1 ( black ) , Rev - erbα ( grey ) and Per2 ( red ) in HIOs ( A ) , kcHIEs ( B ) and bHIEs ( C ) .

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(A-C) mRNA expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIOs (A), kcHIEs (B) and bHIEs (C).


( A ) Representative images of necrotic cell death ( SYTOX orange - red fluorescence ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in control Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f / f - EsrCRE enteroids without tamoxifen treatment . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( D ) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids .

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(A) Representative images of necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids.


( A ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 1μg / mL TcdB in HIOs . Scale bar = 250μm . ( B ) Quantification of necrotic cell death in HIOs . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in kcHIEs . Scale bar = 250μm . ( D ) Quantification of necrotic cell death in kcHIEs . ( E ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in bHIEs . Scale bar = 250μm . ( F ) Quantification of necrotic cell death in bHIEs .

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(A) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.1μg/mL TcdB in HIOs. Scale bar = 250μm. (B) Quantification of necrotic cell death in HIOs. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in kcHIEs. Scale bar = 250μm. (D) Quantification of necrotic cell death in kcHIEs. (E) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in bHIEs. Scale bar = 250μm. (F) Quantification of necrotic cell death in bHIEs.


( A ) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids .

(A) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids.


( B ) Spearman ' s rho for circadian clock and clock - associated genes .

(B) Spearman's rho for circadian clock and clock-associated genes.


( A ) Validation of anti - phasic Rac1 expression in mouse ( mRac1 , red ) and human enteroids ( hRac1 , black ) by qRT - PCR . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1 - KD bHIEs .

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(A) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs.


( C - D ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f / f ; CAGGCre - ERTM without tamoxifen ( C ) or Rac1 f / f ; CAGGCre - ERTM with tamoxifen - treated ( D ) enteroids .

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

(C-D) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.


B . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected WT mice . They are the means ± S . E . M . of results obtained in five mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test .

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

B. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected WT mice. They are the means ± S.E.M. of results obtained in five mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.


C . Top : schemas illustrating the behavioral tasks examined in WT mice . Bottom : the plots represent the discrimination index for the novel object recognition task ( Vehicle N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) , the sociability index ( Vehicle : N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) and the discrimination index for the social discrimination task ( N = 8 for each group ) , measured in each condition . * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index ( exploration of novel object - exploration of familiar object / total object exploration ) equal to zero .

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

C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero.


D . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5 - HT6 - / - mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected 5 - HT6 - / - mice . They are the means ± S . E . M . of results obtained in four mice per group . n . s . p > 0 . 05 , one - way ANOVA followed by Newman - Keuls test .

D. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5-HT6-/- mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected 5-HT6-/- mice. They are the means ± S.E.M. of results obtained in four mice per group. n.s. p > 0.05, one-way ANOVA followed by Newman-Keuls test.


E . Top : schemas illustrating the behavioral tasks in 5 - HT6 - / - mice . Bottom : the plots represent the discrimination index for the novel object recognition test ( discrimination index : 0 . 30 ± 0 . 05 , N = 10 and 0 . 31 ± 0 . 03 , N = 11 , for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , the 3 - chamber social preference test ( sociability index : 0 . 49 ± 0 . 04 , N = 10 and 0 . 48 ± 0 . 07 , N = 11 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) and the social discrimination test ( discrimination index : 0 . 15 ± 0 . 06 , N = 8 and 0 . 21 ± 0 . 06 , N = 8 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , measured in each condition . One - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line corresponds to a discrimination index equal to zero ) .

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

E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).


A - E . The PFC was stained with CB1 receptor ( A1 - D1 , green ) or 5HT6 receptor ( E1 - H1 , green ) , Bassoon ( A2 and E2 , red ) , GAD65 ( B2 and F2 , red ) , PSD - 95 ( C2 and G2 , red ) , or SERT ( D2 and H2 , red ) antibodies . Merge images are also depicted ( A3 - H3 ) . CB1 receptors were mainly co - localized with the presynaptic marker Bassoon ( white arrows , A3 ) , mostly within GABAergic boutons ( white arrows , B3 ) and to a lesser extend with PSD - 95 ( white arrows , C3 ) and SERT ( D3 ) , while 5 - HT6 receptors were mainly co - localized with PSD - 95 ( white arrows , G3 ) . No co - localization of CB1 and 5 - HT6 receptors was detected ( white arrows indicate the 5 - HT6 staining , H3 ) . Scale bar : 10 μm . The scatter plot represents the co - localization analysis for CB1 and 5 - HT6 receptor immunostainings . Graph on the top , Mander ' s split coefficient was used to identify the fraction of CB1 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 66 ± 0 . 02 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 56 ± 0 . 01 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 15 ± 0 . 01 , n = 4 ) or SERT ( Mander ' s coefficient : 0 . 082 ± 0 . 004 , n = 4 ) . Graph on the bottom . Mander ' s split coefficient was used to identify the fraction of 5 - HT6 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 09 ± 0 . 01 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 059 ± 0 . 004 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 58 ± 0 . 01 , n = 4 ) or CB1 receptors ( Mander ' s coefficient : 0 . 0020 ± 0 . 0004 , n = 4 ) . Error bars correspond to the mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. The PFC was stained with CB1 receptor (A1-D1, green) or 5HT6 receptor (E1-H1, green), Bassoon (A2 and E2, red), GAD65 (B2 and F2, red), PSD-95 (C2 and G2, red), or SERT (D2 and H2, red) antibodies. Merge images are also depicted (A3-H3). CB1 receptors were mainly co-localized with the presynaptic marker Bassoon (white arrows, A3), mostly within GABAergic boutons (white arrows, B3) and to a lesser extend with PSD-95 (white arrows, C3) and SERT (D3), while 5-HT6 receptors were mainly co-localized with PSD-95 (white arrows, G3). No co-localization of CB1 and 5-HT6 receptors was detected (white arrows indicate the 5-HT6 staining, H3). Scale bar: 10 μm. The scatter plot represents the co-localization analysis for CB1 and 5-HT6 receptor immunostainings. Graph on the top, Mander's split coefficient was used to identify the fraction of CB1 receptors that co-localizes with Bassoon (Mander's coefficient: 0.66 ± 0.02, n=4), GAD65 (Mander's coefficient: 0.56 ± 0.01, n=4), PSD95 (Mander's coefficient: 0.15 ± 0.01, n=4) or SERT (Mander's coefficient: 0.082 ± 0.004, n=4). Graph on the bottom. Mander's split coefficient was used to identify the fraction of 5-HT6 receptors that co-localizes with Bassoon (Mander's coefficient: 0.09 ± 0.01, n=4), GAD65 (Mander's coefficient: 0.059 ± 0.004, n=4), PSD95 (Mander's coefficient: 0.58 ± 0.01, n=4) or CB1 receptors (Mander's coefficient: 0.0020 ± 0.0004, n=4). Error bars correspond to the mean ± SEM.


B . Top : representative Western blots assessing mTOR activity in PFC are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected mice . They are the means ± S . E . M . of results obtained in six mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test .

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

B. Top: representative Western blots assessing mTOR activity in PFC are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected mice. They are the means ± S.E.M. of results obtained in six mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.


C . The plots represent the discrimination index measured in each condition . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test . The discrimination index for the novel object recognition task is 0 . 34 ± 0 . 06 ( N = 14 ) and - 0 . 08 ± 0 . 07 ( N = 20 ) , for mice injected with Vehicle and THC , respectively , p < 0 . 001 and - 0 . 09 ± 0 . 08 ( N = 21 ) and 0 . 00 ± 0 . 06 ( N = 18 ) , for mice treated with THC + SB258285 and THC + Rapa , respectively , p > 0 . 05 vs . THC - injected mice ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index equal to zero ) .

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

C. The plots represent the discrimination index measured in each condition. *** p < 0.001, one-way ANOVA followed by Bonferroni test. The discrimination index for the novel object recognition task is 0.34 ± 0.06 (N=14) and -0.08 ± 0.07 (N= 20), for mice injected with Vehicle and THC, respectively, p < 0.001 and -0.09 ± 0.08 (N=21) and 0.00 ± 0.06 (N=18), for mice treated with THC+SB258285 and THC+Rapa, respectively, p > 0.05 vs. THC-injected mice (error bars correspond to the mean ± SEM, the dotted line to a discrimination index equal to zero).


B . Left : representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of GABA mIPSC frequency and amplitude measured during the last minute of recording . The mIPSC frequency is : 3 . 2 ± 0 . 4 Hz and 1 . 3 ± 0 . 2 Hz for Vehicle ( n = 9 from N = 4 ) and THC ( n = 7 from N = 4 ) conditions , respectively , p < 0 . 001 and 2 . 5 ± 0 . 2 Hz and 4 . 1 ± 0 . 4 Hz for THC + SB258585 ( n = 8 from N = 4 ) and THC + Rapa ( n = 8 from N = 4 ) conditions , respectively , p < 0 . 01 and p < 0 . 001 vs . THC - injected mice .

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

B. Left: representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of GABA mIPSC frequency and amplitude measured during the last minute of recording. The mIPSC frequency is: 3.2 ± 0.4 Hz and 1.3 ± 0.2 Hz for Vehicle (n=9 from N= 4) and THC (n=7 from N= 4) conditions, respectively, p < 0.001 and 2.5 ± 0.2 Hz and 4.1 ± 0.4 Hz for THC+SB258585 (n=8 from N= 4) and THC+Rapa (n=8 from N= 4) conditions, respectively, p < 0.01 and p < 0.001 vs. THC-injected mice.


C . Left : representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of AMPA mEPSC frequency and amplitude measured during the last minute of recording , n = neurons from 3 to 5 mice per group . The mEPSC frequency is : 4 . 7 ± 0 . 4 Hz for Vehicle ( n = 9 from N = 7 ) and 6 . 8 ± 0 . 4 Hz for THC ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 01and 5 . 3 ± 0 . 5 Hz and 4 . 6 ± 0 , 3 Hz for THC + SB258585 ( n = 10 from N = 6 ) and THC + Rapa ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 05 and p < 0 . 01 vs . THC - injected mice . In B and C , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , one - way ANOVA followed by Newman - Keuls test .

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

C. Left: representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of AMPA mEPSC frequency and amplitude measured during the last minute of recording, n = neurons from 3 to 5 mice per group. The mEPSC frequency is: 4.7 ± 0.4 Hz for Vehicle (n=9 from N= 7) and 6.8 ± 0.4 Hz for THC (n=9 from N= 5) conditions, respectively, p < 0.01and 5.3 ± 0.5 Hz and 4.6 ± 0,3 Hz for THC+SB258585 (n=10 from N= 6) and THC+Rapa (n=9 from N= 5) conditions, respectively, p < 0.05 and p < 0.01 vs. THC-injected mice. In B and C, * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Newman-Keuls test.


A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . A . The resting membrane potential ( RMP ) was determined immediately after whole - cell formation : - 71 . 8 ± 1 . 2 mV and - 60 . 1 ± 1 . 5 mV for Vehicle and THC conditions , respectively , p < 0 . 001 .

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

A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. A. The resting membrane potential (RMP) was determined immediately after whole-cell formation: -71.8 ± 1.2 mV and -60.1 ± 1.5 mV for Vehicle and THC conditions, respectively, p < 0.001.


A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . B . Action potentials were evoked by a ramp current injection with a 10 pA - step for 2 ms and only the first action potential ( AP ) were used to estimate the AP threshold : - 34 . 1 ± 0 . 7 mV and - 39 . 7 ± 0 . 8 mV for Vehicle - and THC - injected mice , respectively , p < 0 . 001 .

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

A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. B. Action potentials were evoked by a ramp current injection with a 10 pA-step for 2 ms and only the first action potential (AP) were used to estimate the AP threshold: -34.1 ± 0.7 mV and -39.7 ± 0.8 mV for Vehicle- and THC-injected mice, respectively, p < 0.001.


A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . C . The rheobase represents the minimal current required to induce neuronal firing , it was lowered in THC condition : 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions , respectively , p < 0 . 001 .

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

A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. C. The rheobase represents the minimal current required to induce neuronal firing, it was lowered in THC condition: 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions, respectively, p < 0.001.


A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . D . The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs .

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

A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. D. The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs.


E . top : Voltage sag in response to hyperpolarizing current injection ( 50 pA - increments , from - 400 to 0 pA ) in PFC pyramidal neurons from mice injected with vehicle ( Veh , n = 10 from N = 5 ) , THC ( n = 11 from N = 5 ) , THC + SB258585 ( THC / SB , n = 7 from N = 4 ) or THC + Rapa ( THC / Rapa , n = 7 from N = 4 ) during adolescence . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test , vs . THC - injected mice . Errors bars correspond to the mean ± SEM . Voltage sag is indicated by arrowheads .

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

E. top: Voltage sag in response to hyperpolarizing current injection (50 pA-increments, from -400 to 0 pA) in PFC pyramidal neurons from mice injected with vehicle (Veh, n= 10 from N=5), THC (n = 11 from N=5), THC+SB258585 (THC/SB, n=7 from N=4) or THC+Rapa (THC/Rapa, n=7 from N=4) during adolescence. *** p < 0.001, one-way ANOVA followed by Bonferroni test, vs. THC-injected mice. Errors bars correspond to the mean ± SEM. Voltage sag is indicated by arrowheads.


F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( F ) resting potential membrane : THC + ZD : - 74 . 14 ± 1 . 70 mV , p < 0 . 001 vs . THC condition , THC + DDOA : - 70 . 47 ± 1 . 19 mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 63 . 10 ± 0 . 81 mV , p < 0 . 001 vs . THC condition ,

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

F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (F) resting potential membrane: THC + ZD: -74.14 ± 1.70 mV, p < 0.001 vs. THC condition, THC + DDOA: -70.47 ± 1.19 mV, p < 0.001 vs. THC condition, THC + ODQ: -63.10 ± 0.81 mV, p < 0.001 vs. THC condition,


F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( G ) AP threshold : THC + ZD : - 33 . 59 ± 2 . 96 mV , p < 0 . 01 vs . THC condition , THC + DDOA : - 34 . 28 ± 0 . 98mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 36 . 84 ± 0 . 79 mV , p < 0 . 001 vs . THC

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

F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (G) AP threshold: THC + ZD: -33.59 ± 2.96 mV, p < 0.01 vs. THC condition, THC + DDOA: -34.28 ± 0.98mV, p < 0.001 vs. THC condition, THC + ODQ: -36.84 ± 0.79 mV, p < 0.001 vs. THC condition


F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( H ) rheobase : THC + ZD : 806 ± 63 pA , p < 0 . 001 vs . THC condition , THC + DDOA : 607 ± 43 pA , p < 0 . 001 vs . THC condition , THC + ODQ : 422 ± 34 pA , p < 0 . 001 vs . THC

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

F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (H) rheobase: THC + ZD: 806 ± 63 pA, p < 0.001 vs. THC condition, THC + DDOA: 607 ± 43 pA, p < 0.001 vs. THC condition, THC + ODQ: 422 ± 34 pA, p < 0.001 vs. THC condition


F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( I ) firing rate . Note that Vehicle - and THC - injected mice are the same as those used in experiments illustrated on Figure 5A - D . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , n . s . p > 0 . 05 one - way ANOVA followed by Bonferroni test .

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

F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (I) firing rate. Note that Vehicle- and THC-injected mice are the same as those used in experiments illustrated on Figure 5A-D. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s. p > 0.05 one-way ANOVA followed by Bonferroni test.


A . In left , normalized peak amplitudes of isolated AMPA EPSCs recorded at - 60 mV , before and after pairing protocol , are illustrated . Representative traces of AMPA EPSCs before ( 1 ) or after ( 2 ) the pairing protocol are also illustrated ( right panel ) for each experimental condition : Vehicle - injected mice ( white circles , n = 3 from N = 3 ) , THC - injected mice ( green circles , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black circles , n = 3 from N = 3 ) . Errors bars correspond to mean ± SEM .

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

A. In left, normalized peak amplitudes of isolated AMPA EPSCs recorded at -60 mV, before and after pairing protocol, are illustrated. Representative traces of AMPA EPSCs before (1) or after (2) the pairing protocol are also illustrated (right panel) for each experimental condition: Vehicle-injected mice (white circles, n=3 from N=3), THC-injected mice (green circles, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black circles, n=3 from N=3). Errors bars correspond to mean ± SEM.


B . The histogram represents the means ± SEM of AMPA EPSCs in % of baseline , measured during the last 5 min of baseline or the last 5 min of recording , for each experimental condition : vehicle - injected mice ( white bar , n = 3 from N = 3 ) , THC - injected mice ( green bar , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black bar , n = 3 from N = 3 ) , n . s . p > 0 . 05 , * p < 0 . 05 , one - way ANOVA followed by Tukey test .

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

B. The histogram represents the means ± SEM of AMPA EPSCs in % of baseline, measured during the last 5 min of baseline or the last 5 min of recording, for each experimental condition: vehicle-injected mice (white bar, n=3 from N=3), THC-injected mice (green bar, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black bar, n=3 from N=3), n.s. p > 0.05, * p< 0.05, one-way ANOVA followed by Tukey test.


( A ) Representative kymographs showing tetramethylrhodamine ( TMR ) - labeled DDB movement on each type of recombinant MT lattice , as indicated . Scale bars , 5 μm and 25 sec . Pink arrowhead marks a diffusive DDB complex . See also Movie EV1 . ( B ) Quantification of DDB velocity on MT lattices lacking each of the tubulin - CTTs ( n ≥ 100 DDB complexes per condition from at least three independent experiments ) . No statistical difference was observed between WT and mutant MTs ( P ≥ 0 . 1 , unpaired t test ) ( C - D ) Quantification of the number of processive ( C ) , or diffusive ( D ) DDB complexes relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Processive ( unidirectional motion exceeding 0 . 5 μm ) and diffusive ( back - and - forth motion ) events were scored in a field of view and then divided by the lengths of MTs and time , thus normalizing to a value of number of motility events per μm MT per sec . Error bars SD .

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

(A) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD.


( E - F ) TIRF images of 1 nM TMR - labeled GST - hDyn ( 2 mM ATP ) ( E ) , or 0 . 3 nM TMR - labeled p150 ( F ) molecules ( green ) bound to either Δα - CTT ( top ) , or Δβ - CTT MTs ( bottom ) . Pink arrows show mutant MTs in both the raw , and SD map images , highlighting differences in binding between WT and mutant MTs that are apparent in the SD map . Right , standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities ( see Methods ) . Scale bars are 5 μm . ( G ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . Image shows first frame from time series . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD . P value ( unpaired t test ) of Δα - CTT vs . Δβ - CTT MTs = 0 . 5208 for GST - hDyn , and ≤ 0 . 0001 for p150 .

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

(E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.


( A ) TIRF image of TMR - labeled DDB molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . A standard deviation map of the whole time sequence is shown , revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs ( pink arrow ) . Representative kymographs of DDB movement are shown from each type of MT present in the same chamber . Scale bars are 5 μm and 25 sec . See also Movie EV2 . ( B - C ) Quantification of the number of processive ( B ) or diffusive ( C ) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber ( n ≥ 20 MTs quantified for each condition from three independent experiments ) . Error bars represent SD . Data from Δα - CTT MTs ( Fig . 1 ) are re - plotted for comparison . ( D ) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs . Data points from two independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown.


( E ) TIRF images of 0 . 3 nM TMR - labeled p150 ( top ) or 1 nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown . Scale bars are 5 μm and 25 sec . ( F ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT . ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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

(E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Immunoblot of brain tubulin with or without carboxypeptidase A ( CPA ) . Note CPA specifically removes the α - tubulin C - terminal tyrosine without affecting other PTMs .

O O O O O O O O O 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) Immunoblot of brain tubulin with or without carboxypeptidase A (CPA). Note CPA specifically removes the α-tubulin C-terminal tyrosine without affecting other PTMs.


( B ) TIRF image of TMR - labeled DDB molecules ( green ) on either WT ( red ) or CPA - treated ( blue ) pig brain MTs . Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec . ( C ) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA - treated MTs , relative to WT MTs in the same chamber ( n ≥ 7 MTs of each type quantified from at least three independent experiments ) . Error bars represent SD .

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

(B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD.


( D ) TIRF images of 0 . 3nM TMR - labeled p150 ( top ) or 1nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either WT ( red ) , or CPA - treated MTs ( blue ) . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec for p150 , and 5 μm and 12 sec for GST - hDyn ( E ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2mM ATP ) or p150 bound to CPA - treated MTs relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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

(D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Schematic of the p150 constructs used . The p135 isoform lacks the CAP - Gly domain , but retains part of the adjacent basic domain ( red ) and coiled - coil domains ( blue ) . Right , Coomassie blue stained gel of the purified p150 and p135 proteins . Molecular weight markers are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated.


( B ) TIRF image of either p150 ( 0 . 3 nM ) , or p135 ( 0 . 3 or 6 nM ) bound to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Note : no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform . Proteins were visualized by fluorescence from the GFP tag fused to each construct .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TIRF image of either p150 (0.3 nM), or p135 (0.3 or 6 nM) bound to either WT (red), or CPA-treated porcine MTs (blue). Note: no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform. Proteins were visualized by fluorescence from the GFP tag fused to each construct.