A - C . Immunoblot analyses of Hog1 phosphorylation . Yeast strains ( A ) KT235 ; ( B ) KT209 ; ( C ) KT234 were stimulated with indicated concentrations of NaCl for 10 min , phosphorylated Hog1 ( Hog1 - P ) total Hog1 in lysates were detected by immunoblotting . relevant genotypes of strains are indicated in top row of each panel , are schematically shown in diagrams at left . second row from top indicates genes carried by a single - copy plasmid that are expressed from their own promoters . vec , vector ; WT , wild - type .

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A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.


D . Phos - tag band - shift analyses of Hog1 phosphorylation . yeast KT234 carrying single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was stimulated with indicated concentrations of NaCl for 5 min .

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D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.


E . Comparison of NaCl dose - responses of Hog1 activation by constitutively - active Ste11 - Q301P constitutively - active Pbs2 - DD . Phos - tag band - shift assays were independently repeated three times , average values were plotted . Data information : ( E ) Error bars are ( n = 3 ) .

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E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays were independently repeated three times, and the average values were plotted. Data information: (E) Error bars are SEM (n=3).


C . Immunoblot analyses of Hog1 phosphorylation . yeast KT235 was transformed with pRS416 - FLAG - Hog1 ( WT ) or its indicated deletion derivatives . FLAG - Hog1 was immunoprecipitated ( IP ) , immunoblotted ( IB ) with anti - phospho - p38 ( for Hog1 - P ; upper panel ) or anti - FLAG ( for total FLAG - Hog1 ; lower panel )

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C. Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416-FLAG-Hog1 (WT) or its indicated deletion derivatives. FLAG-Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti-phospho-p38 (for Hog1-P; upper panel) or anti-FLAG (for total FLAG-Hog1; lower panel)


D - G . Phos - tag band - shift of Hog1 phosphorylation . Yeast ( D - E ) KT235 or ( F - G ) KT290 carrying single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was transformed with either pRS416 - Hog1 ( WT ) or its indicated mutant derivatives , was treated with indicated concentrations of NaCl for 5 min . ( D ) ( F ) show typical results , ( E ) ( G ) summarizes averages of three independent experiments . Data information : ( E , G Error bars are ( n = 3 ) .

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D-G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D-E) KT235 or (F-G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives, and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarizes the averages of three independent experiments. Data information: (E, G Error bars are SEM (n=3).


H . Phos - tag band - shift of Hog1 phosphorylation . yeast FP4 was transformed with single - copy expression plasmid pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - ΔL16 , was treated with indicated concentrations of NaCl for 5 min . averages of three independent experiments are shown . Data information : Error bars are ( n = 3 ) .

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H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16, and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: Error bars are SEM (n=3).


A . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . yeast FP4 ( hog1Δ ) was transformed with single - copy expression plasmid pRS416 - Hog1 - ΔL16 , was stimulated with indicated concentrations of NaCl for 5 min . B . Averages of three independent experiments from A were plotted . Results for Hog1 - WT ( from Fig 1I ) are included for comparison . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min. B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


C - D . yeast KT259 ( ste11Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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C-D. the yeast strain KT259 (ste11Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


E - F . yeast KY523 ( ssk2 / 22Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are ( n = 3 ) .

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E-F. the yeast strain KY523 (ssk2/22Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


Detection of Pbs2 phosphorylation at S514 yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extract , phosphorylated Pbs2 was analyzed by ( A ) Phos - tag band - shift In ( A ) , positions of Pbs2 - HA phosphorylated ( S514 - P ) unphosphorylated ( S514 - OH ) at S514 are indicated .

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Detection of Pbs2 phosphorylation at S514 The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (A) Phos-tag band-shift assay In (A), the positions of Pbs2-HA phosphorylated (S514-P) and unphosphorylated (S514-OH) at S514 are indicated.


Detection of Pbs2 phosphorylation at T518 . yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extract , phosphorylated Pbs2 was analyzed by ( B ) anti - phospho - T518 immunoblotting .

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Detection of Pbs2 phosphorylation at T518. The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (B) anti-phospho-T518 immunoblotting.


C - F . NaCl dose - response analyses of Pbs2 phosphorylation . ( C - D ) KT005 ( ste11Δ pbs2Δ ) ( E - F ) TM280 ( ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , were treated with indicated concentrations of NaCl for 5 min . ( C E ) S514 phosphorylation was analyzed using Phos - tag band - shift . PC ; Positive Control . ( D F ) T518 phosphorylation was analyzed using anti - phospho - T518 immunoblotting . G - H . Average values of three independent experiments from ( C - D ) ( E - F ) , respectively , were plotted . AU , arbitrary unit . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are ( n = 3 ) .

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C-F. NaCl dose-response analyses of Pbs2 phosphorylation. (C-D) KT005 (ste11Δ pbs2Δ) and (E-F) TM280 (ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. (C and E) S514 phosphorylation was analyzed using the Phos-tag band-shift assay. PC; Positive Control. (D and F) T518 phosphorylation was analyzed using anti-phospho-T518 immunoblotting. G-H. Average values of three independent experiments from (C-D) and (E-F), respectively, were plotted. AU, arbitrary unit. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


I . Detection of di - phosphorylated Pbs2 . KT005 ( ste11Δ pbs2Δ ) transformed with YCplac22I ' - Pbs2 - HA was treated with indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from extracts , subjected to Phos - tag SDS - PAGE . Blots of these gels were probed with ( upper panel ) anti - phospho - T518 or ( lower panel ) anti - HA . J . Same as in ( I ) , except that yeast KT003 ( pbs2Δ ) was used .

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I. Detection of di-phosphorylated Pbs2. KT005 (ste11Δ pbs2Δ) transformed with YCplac22I'-Pbs2-HA was treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extracts, and subjected to Phos-tag SDS-PAGE. Blots of these gels were probed with (upper panel) anti-phospho-T518 or (lower panel) anti-HA. J. Same as in (I), except that the yeast strain KT003 (pbs2Δ) was used.


B . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . KT005 ( ste11Δ pbs2Δ ) was transformed with YCplac22I ' - Pbs2 - HA ( WT ) or its indicated mutant derivatives . were stimulated with ( + ) or without ( - ) 0 . 6 M NaCl for 5 min .

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B. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. KT005 (ste11Δ pbs2Δ) was transformed with YCplac22I'-Pbs2 -HA (WT) or its indicated mutant derivatives. Cells were stimulated with (+) or without (-) 0.6 M NaCl for 5 min.


C - F . Phos - tag band - shift analyses of Hog1 phosphorylation . KT291 ( ste11Δ pbs2Δ hog1Δ ) was transformed with ( C D ) pRS416 - Hog1 or ( E F ) pRS416 - Hog1 - ΔL16 , together with ( C E ) YCplac22I ' - Pbs2 - S514A - HA or ( D F ) YCplac22I ' - Pbs2 - T518A - HA . were stimulated with indicated concentrations of NaCl for 5 min . G - H . Average values of three independent experiments from ( C E ) ( D F ) , respectively , are plotted . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are ( n = 3 ) .

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C-F. Phos-tag band-shift analyses of Hog1 phosphorylation. KT291 (ste11Δ pbs2Δ hog1Δ) was transformed with (C and D) pRS416-Hog1 or (E and F) pRS416-Hog1-ΔL16, together with (C and E) YCplac22I'-Pbs2-S514A-HA or (D and F) YCplac22I'-Pbs2-T518A-HA. Cells were stimulated with the indicated concentrations of NaCl for 5 min. G-H. Average values of three independent experiments from (C and E) and (D and F), respectively, are plotted. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


A Phos - tag band - shift of Hog1 phosphorylation . KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives . extracts were prepared from fresh cultures without applying osmostress . For each HOG1 mutant plasmid , three independent cultures were assayed . Data information : Error bars are ( n = 3 ) .

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A Phos-tag band-shift assay of Hog1 phosphorylation. KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives. Cell extracts were prepared from fresh cultures without applying osmostress. For each HOG1 mutant plasmid, three independent cultures were assayed. Data information: Error bars are SEM (n=3).


B . yeast strains KT260 ( ssk2 / 22Δ ste11Δ hog1Δ ) , KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) , KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) carrying YCplac22I ' - Pbs2 - DD , were used . Data information : Error bars are ( n = 3 ) .

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B. the yeast strains KT260 (ssk2/22Δ ste11Δ hog1Δ), KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P), and KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) carrying YCplac22I'-Pbs2-DD, were used. Data information: Error bars are SEM (n=3).


C . KT250 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - WT ) KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) were transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N149H D162G ) together with pRS414 - 8xCRE - lacZ . Expression of Hog1 reporter gene 8xCRE - lacZ in absence of osmostress was assayed . Data information : ( C ) Error bars are ( n = 4 ) .

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C. KT250 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-WT) and KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) were transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N149H D162G) together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: (C) Error bars are SEM (n=4).


D . KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives together with pRS414 - 8xCRE - lacZ . Expression of Hog1 reporter gene 8xCRE - lacZ in absence of osmostress was assayed . Data information : Error bars are ( n = 3 ) .

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D. KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: Error bars are SEM (n=3).


E - F . Phos - tag band - shift of Hog1 phosphorylation . Yeast strains of indicated genotypes ( shown below graph ) were transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives ( shown inside graph ) . used : ( E ) FP4 , KT259 , KT260 , KT292 , KY523 ; ( F ) KY523 , KT293 , KT294 , KT295 . N / H , N149H ; D / G , D162G . Data information : Error bars are ( n = 3 ) .

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E-F. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strains of the indicated genotypes (shown below the graph) were transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives (shown inside the graph). Strain used: (E) FP4, KT259, KT260, KT292, and KY523; and (F) KY523, KT293, KT294, and KT295. N/H, N149H; D/G, D162G. Data information: Error bars are SEM (n=3).


A . KT299 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ ) was exposed to indicated concentrations of α - factor for 15 min in absence of osmostress . Phosphorylation of Fus3 Kss1 was detected by immunoblotting .

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A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.


B . KT299 ( MATa ssk2 / 22Δ hkr1Δ msb2Δ ) was exposed to indicated concentrations of α - factor for 15 min in presence or absence of 0 . 8 M NaCl , Hog1 phosphorylation was determined using Phos - tag band - shift . Data information : ( B ) Representative results from three independent experiments .

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B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Data information: (B) Representative results from three independent experiments.


C . Average values of four independent experiments were plotted . Data information : Error bars are ( C ) n =

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C. Average values of four independent experiments were plotted. Data information: Error bars are SEM (C) n=4


D . HM06 - 1 ( MATa ΔS / O / H / M ssk2 / 22Δ ) was exposed to indicated concentrations ( log scale ) of α - factor for 15 min in presence or absence of 1 . 0 M NaCl , Hog1 phosphorylation was determined using Phos - tag band - shift . Average values of three or more independent experiments were plotted . Data information : Error bars are : ( D ) , n = 3 or

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D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted. Data information: Error bars are SEM: (D), n=3 or more


E . KT306 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ hog1Δ ) was transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N / H D / G ) , was exposed to 10 μM α - factor for indicated time in absence of osmostress , Hog1 phosphorylation was determined using Phos - tag band - shift . Average of three independent experiments are plotted . Data information : Error bars are ( E ) n = 3 .

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E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted. Data information: Error bars are SEM (E) n=3.


G . Typical FRET ( YFP / CFP ) showing p38 activation . HeLa carrying p38 reporter PerKy - p38 ( Tomida et al . , 2015 ) were stably transfected with an expression vector for indicated p38α mutant . FRET was performed as described in Materials Methods . H . Distribution of p38 activity in from sets ( a ) - ( d ) in ( G ) . Data information : ( G ) Scale bars : 20 μm . ( H ) Statistics , Student ' s two - t - .

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G. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy-p38 (Tomida et al., 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods. H. Distribution of p38 activity in individual cells from sets (a) - (d) in (G). Data information: (G) Scale bars: 20 μm. (H) Statistics, Student's two-tailed t-test.


A . An example of time - course experiments for osmostress - induced Hog1 phosphorylation . yeast KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) was stimulated with 1 . 0 M NaCl for indicated times , of phosphorylated Hog1 ( Hog1 - P ) was determined using a Phos - tag band - shift .

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A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay.


Compilations of time - courses of osmostress - induced Hog1 activation in ( B ) KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) , For clarity , time - course curves are shown in two panels for lower higher ranges of NaCl concentrations . Data information : For each data point , n = 1 or more .

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Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. Data information: For each data point, n=1 or more.


Compilations of time - courses of osmostress - induced Hog1 activation in ( C ) TM257 ( ssk2 / 22Δ ) , ( D ) TM142 ( wild - type ) . For clarity , time - course curves are shown in two panels for lower higher ranges of NaCl concentrations . color chart below ( D ) indicates concentrations of NaCl used . Data information : For each data point , n = 1 or more .

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Compilations of the time-courses of osmostress-induced Hog1 activation in (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used. Data information: For each data point, n=1 or more.


E . Effects of Hog1 kinase activity osmostress - induced glycerol accumulation on time - courses of Hog1 activation . were stimulated with 0 . 4 M NaCl ( left panel ) or 1 . 6 M NaCl ( right panel ) for indicated times , of phosphorylated Hog1 was determined using Phos - tag band - shift . Strains used were : TM142 ( WT ) , KT254 ( gpd1Δ gpd2Δ stl1Δ ) , TM232 ( hog1Δ ) carrying pRS416 - HOG1 - K52S K53N . K / S K / N , K52S K53N ; gpd1 / 2 , gpd1Δ gpd2Δ . Data information : ( E ) Error bars are ( n = 3 or more ) .

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E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ. Data information: (E) Error bars are SEM (n=3 or more).


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

(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 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 raw , SD map , highlighting differences in binding between WT mutant MTs that are apparent in SD map . Right , standard deviation maps from an entire time reveal ensemble binding dissociation events that lead to variations in pixel intensities ( see Methods ) . Scale bars are 5 μm . ( G ) of mean ( arbitrary units ) per μm MT for GST - hDyn ( 2 mM ATP ) or p150 bound to indicated mutant MT relative to WT MTs in same chamber . Image shows first frame from time series . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD . P value ( unpaired t ) of Δα - CTT vs . Δβ - CTT MTs = 0 . 5208 for GST - hDyn , ≤ 0 . 0001 for p150 .

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(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 whole time is shown , revealing 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 same chamber . Scale bars are 5 μm 25 sec . See also Movie EV2 . ( B - C ) of number of processive ( B ) or diffusive ( C ) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in 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 ) of frequency of processive DDB molecules as a function of amount of tyrosinated tubulin incorporated into lattice normalized to fully tyrosinated MTs . Data points from two independent experiments are shown .

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(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 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 binding over an entire time kymographs from either tyrosinated or detyrosinated MTs are shown . Scale bars are 5 μm 25 sec . ( F ) of mean ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2 mM ATP ) or p150 bound to indicated mutant MT relative to WT MTs in same chamber . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT . ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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(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 α - tubulin C - terminal tyrosine without affecting other PTMs .

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(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 entire time shows DDB preference for WT MTs kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm 25 sec . ( C ) of number of processive DDB complexes per μm MT per sec on CPA - treated MTs , relative to WT MTs in same chamber ( n ≥ 7 MTs of each type quantified from at least three independent experiments ) . Error bars represent SD .

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(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 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 binding over an entire time kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm 25 sec for p150 , 5 μm 12 sec for GST - hDyn ( E ) of mean ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2mM ATP ) or p150 bound to CPA - treated MTs relative to WT MTs in same chamber . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

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

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(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 lower binding affinity of this isoform . were visualized by from GFP tag fused to each construct .

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(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.


( C ) Still from a TIRF time - series of p135 ( green ) binding to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Right , standard deviation map of p135 binding shows no preference for either type of MT , of mean ( arbitrary units ) per μm MT for p135 bound to CPA - treated MTs relative to WT MTs in same chamber ( n ≥ 10 MTs quantified for each condition from three independent experiments ) . Scale bars are 5 μm .

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(C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.


( B ) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated ( red ) detyrosinated ( blue ) sections . Most TMR - labeled DDB molecules ( green ) move processively through annealed junction between two types of MT lattice ( 65 % , n = 836 processive complexes , 65 MT - MT junctions , three independent experiments ) . Diffusive DDB molecules ( arrow ) are primarily observed on tyrosinated section of MT do not diffuse across boundary . Right , video frames showing a processive DDB ( pink arrowhead ) moving across junction ( yellow arrowhead ) of tyrosinated detryosinated MT . ( + ) ( - ) signs denote MT polarity inferred from directionality of DDB movement . Scale bars are 5 μm 25 sec . See also Movie EV3 .

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(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.


( C ) TIRF of low ( 0 . 3 nM ) high ( 10 nM ) concentrations of TMR - labeled p150 ( green ) bound to chimeric yeast MTs containing tyrosinated ( red ) detyrosinated ( blue ) sections of MT . Representative kymographs below show diffusive behavior of p150 molecules on MT . Note that p150 rarely diffuses into detyrosinated section of MT , even at high concentrations . Scale bars are 5 μm 10 sec .

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(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.


( D - E ) TIRFimages kymographs of annealed WT CPA - treated porcineMTs ( D ) , or annealed WT MTs ( E ) . majority of DDB complexes ( 78 % , n = 960 processive complexes , 45 MT - MT junctions , three independent experiments ) move processively across boundary between WT CPA - MTs ( D ) . Note TMR - labeled DDB complexes ( green ) in ( E ) traverse annealed boundary without pausing ( 93 % , n = 677 processive complexes , 45 MT - MT junctions , three independent experiments ) , suggesting that annealed junction does not affect DDB motility .

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(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.


( F ) TIRF image kymograph from a chimeric MT composed of WT ( red ) subtilisin - treated porcine MTs ( blue ) . Note some DDB complexes ( pink arrows ) traverse boundary continue moving along subtilisin - treated section of MT ( 41 % , n = 709 processive complexes , 56 MT - MT junctions , 3 independent experiments ) . scale bars in D - F are 5 μm 25 sec .

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(F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.


( A ) UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies conditioned on yeast paste had diffuse GFP - mCherry - DrAtg8a in midstage egg chambers . ( B ) Nondegenerating midstage egg chambers from starved flies contained autophagosomes ( yellow ) autolysosomes ( red ) . ( C D ) Egg chambers early in degeneration process showed follicle that take up portions of nurse cytoplasm ( C ) followed by condensation fragmentation of nurse nuclei further uptake of nurse cytoplasm into follicle ( D ) . ( E ) Late stage degenerating egg chambers lose GFP fluoresce red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies conditioned on yeast paste had diffuse GFP-mCherry-DrAtg8a staining in midstage egg chambers. (B) Nondegenerating midstage egg chambers from starved flies contained autophagosomes (yellow) and autolysosomes (red). (C and D) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm (C) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells (D). (E) Late stage degenerating egg chambers lose all GFP staining and fluoresce red.


( F ) Fed Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed diffuse GFP - mCherry - DrAtg8a in germline . ( G ) Starved Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers . ( H I ) Fed flies overexpressing truncated Dcp - 1 ( tDcp - 1 ) in germline showed increased autophagosomes autolysosomes in nondegenerating midstage egg chambers ( H ) also contained degenerating midstage egg chambers that lose GFP fluoresce red ( I ) . Bars : ( main ) 25 µm ; ( insets ) 12 . 5 µm . Insets in A - I show diffuse of Atg8a or autophagosomes autolysosomes . ( J K ) percentages of autolysosomes ( autolysosomes / total autophagic structures ) were manually calculated in at least eight egg chambers for each genotype as indicated . Error bars represent means ± SD . Statistical testing was performed using one - way ANOVA with a Dunnet post ( * * * * , P < 0 . 0001 ) or a two - Student ' s t ( * * , P < 0 . 005 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fed Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed diffuse GFP-mCherry-DrAtg8a staining in the germline. (G) Starved Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers. (H and I) Fed flies overexpressing truncated Dcp-1 (tDcp-1) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers (H) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red (I). Bars: (main images) 25 µm; (insets) 12.5 µm. Insets in A-I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes. (J and K) The percentages of autolysosomes (autolysosomes/total autophagic structures) were manually calculated in at least eight egg chambers for each genotype as indicated. Error bars represent the means ± SD. Statistical testing was performed using one-way ANOVA with a Dunnet post test (****, P < 0.0001) or a two-tailed Student's t test (**, P < 0.005).


( A ) Drosophila S2 stably expressing GFP - RFP - Atg8a showed an increase in of containing two or more autolysosomes after starvation , which was blocked after Bafilomycin A1 ( BafA1 ) treatment . At least 50 were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post ( * * , P < 0 . 01 ) . ( B ) Representative of S2 - GFP - RFP - Atg8a after indicated treatments .

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

(A) Drosophila S2 cells stably expressing GFP-RFP-Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation, which was blocked after Bafilomycin A1 (BafA1) treatment. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01). (B) Representative images of S2-GFP-RFP-Atg8a cells after the indicated treatments.


( C ) S2 - GFP - RFP - Atg8a were treated with dsRNAs subjected to fed or starvation conditions as indicated . At least 50 were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post ( * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ) . ( D ) Representative of S2 - GFP - RFP - Atg8a after indicated RNAi treatments . Error bars represent means ± SD . Bars , 10 µm .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S2-GFP-RFP-Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Representative images of S2-GFP-RFP-Atg8a cells after the indicated RNAi treatments. Error bars represent the means ± SD. Bars, 10 µm.


( A ) Nondegenerating midstage egg chambers from fed w1118flies showed Ref ( 2 ) P in follicle nurse . ( B ) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref ( 2 ) P . ( D ) Starved Atg7flies showed an accumulation of Ref ( 2 ) P in follicle nurse of degenerating midstage egg chambers . ( E ) Starved Dcp - 1Prev1flies contained increased of Ref ( 2 ) P in degenerating midstage egg chambers .

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

(A) Nondegenerating midstage egg chambers from fed w1118flies showed Ref(2)P staining in follicle cells and nurse cells. (B) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref(2)P.(D) Starved Atg7flies showed an accumulation of Ref(2)P in the follicle cells and nurse cells of degenerating midstage egg chambers. (E) Starved Dcp-1Prev1flies contained increased levels of Ref(2)P in degenerating midstage egg chambers.


( C ) A representative Western blot of ovaries from w1118 Atg7d77 / d14 flies subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P relative to actin . Graph represents ± SD from five independent experiments ( n = 5 ) . Statistical significance was determined using a two - Student ' s t . * , P = 0 . 004 for fed samples ; * , P = 0 . 008 for starved samples .

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

(C) A representative Western blot of ovaries from w1118 and Atg7d77/d14 flies subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test. *, P = 0.004 for fed samples; *, P = 0.008 for starved samples.


( F ) A representative Western blot of ovaries from w1118 Dcp - 1Prev1 flies that were subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P relative to actin . Graph represents ± SD from five independent experiments ( n = 5 )

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

(F) A representative Western blot of ovaries from w1118 and Dcp-1Prev1 flies that were subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5)


( A B ) l ( 2 ) mbn were labeled with antibodies to Dcp - 1 ATPsyn - α ( A ) or MitoTracker red ( MTR ; B ) . Merged show colocalization between Dcp - 1 mitochondria . Boxes represent zoomed . Bars : ( main ) 5 µm ; ( zoomed ) 1 . 25 µm .

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

(A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm.


( C ) Western blot from l ( 2 ) mbn subjected to nutrient - rich or starvation conditions for 6 h . were separated into ( C ) mitochondrial enriched ( M ) fractions .

O O O O O O O O O O O O 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) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions.


( D ) Ovaries from fed w1118 flies were separated into - enriched fractions probed with antibodies to VDAC , Tubulin , ATPsyn - α , Dcp - 1 .

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

(D) Ovaries from fed w1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1.


( E F ) Intact lysed mitochondria ( E ) or intact mitochondria isolated from l ( 2 ) mbn ( F ) were treated with proteinase K ( PK ) . effects of proteinase K treatment were assessed by antibodies to VDAC , ATPsyn - α , Pink1 , Dcp - 1 .

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

(E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1.


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


( G ) Control Dcp - 1 RNAi - treated were subjected to nutrient - rich or starvation conditions stained with NAO . Mean was measured by flow cytometry . Graph represents ± of three independent experiments ( n = 3 ) .

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

(G) Control and Dcp-1 RNAi-treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments (n = 3).


( A ) l ( 2 ) mbn were labeled with ATPsyn - α , mitochondrial was scored as fragmented , normal , or elongated . ( B ) were treated with control or Dcp - 1 dsRNA subjected to nutrient - rich media or 1 h of starvation . Quantifications represent of with elongated mitochondria divided by total number of examined . At least 100 were examined manually in three independent experiments ( n = 3 ) . Error bars represent mean ± SD . Statistical significance was determined using one - way ANOVA with a Bonferroni post ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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

(A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments (n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P < 0.05; **, P < 0.01).


( C ) Mitochondrial targeted GFP ( mitoGFP ) was expressed in germline using nosGAL4 driver . shows mitoGFP , Armadillo , . ( D ) Mitochondria were scored as healthy ( H ) , clustered ( C ) , or elongated overly connected ( E ) . of mitochondria from fed UASp - mitoGFP / + ; nosGAL4 / + flies were scored as healthy . n = 15 egg chambers manually scored . ( E ) mitoGFP was expressed in Dcp - 1Prev1 flies using nosGAL4 driver . ( F ) 54 % of egg chambers from UASp - mitoGFP / + ; Dcp - 1Prev1 / Dcp - 1Prev1 ; nosGAL4 / + flies contained elongated mitochondria , 39 % contained mitochondria that were scored as healthy , 7 % contained clustered mitochondria . n = 28 egg chambers manually scored .

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

(C) Mitochondrial targeted GFP (mitoGFP) was expressed in the germline using the nosGAL4 driver. Staining shows mitoGFP, Armadillo, and DNA. (D) Mitochondria were scored as healthy (H), clustered (C), or elongated and overly connected (E). All of mitochondria from fed UASp-mitoGFP/+;nosGAL4/+ flies were scored as healthy. n = 15 egg chambers manually scored. (E) mitoGFP was expressed in Dcp-1Prev1 flies using the nosGAL4 driver. (F) 54% of egg chambers from UASp-mitoGFP/+;Dcp-1Prev1/Dcp-1Prev1;nosGAL4/+ flies contained elongated mitochondria, 39% contained mitochondria that were scored as healthy, and 7% contained clustered mitochondria. n = 28 egg chambers manually scored.


( A ) Total ATP were measured in l ( 2 ) mbn treated with control or Dcp - 1 dsRNA . Data represent ± of three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Students t ( * , P = 0 . 02 ) .

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

(A) Total cellular ATP levels were measured in l(2)mbn cells treated with control or Dcp-1 dsRNA. Data represent ± SEM of three independent experiments (n = 3). Statistical significance was determined using a two-tailed Students t test (*, P = 0.02).


( B ) Total ATP were measured in ovaries from fed or starved w1118 Dcp - 1Prev1 flies . Data represent ± of five independent experiments ( n = 5 ) . Statistical significance was determined using a two - Student ' s t ( * * , P = 0 . 014 ; * * * , P < 0 . 001 ) .

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

(B) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001).


( C ) Fed or starved w1118 Dcp - 1Prev1 flies were treated with DMSO or 25 µg / ml oligomycin A , Ref ( 2 ) P were assessed by immunoblot . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graph represents ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P = 0 . 02 ) .

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

(C) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2)P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02).


( D E ) Control UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies ( D ) Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies ( E ) were subjected to starvation conditions supplemented with DMSO or 25 µg / ml oligomycin A . Bars , 25 µm . Quantifications show of autolysosomes ( autolysosomes / total autophagic structures ) . At least eight egg chambers were manually quantitated per genotype per condition ( n = 8 ) . Statistical testing was determined using a two - Student ' s t . * * * , P = 0 . 0002 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.


( A ) A representative Western blot showing SesB ATPsyn - α from fed or starved l ( 2 ) mbn treated with control or Dcp - 1 RNAi . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P < 0 . 05 ) .

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

(A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( B ) A representative Western blot of ovaries from fed or starved w1118 or Dcp - 1Prev1 flies . SesB ATPsyn - α were detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P < 0 . 05 ) .

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

(B) A representative Western blot of ovaries from fed or starved w1118 or Dcp-1Prev1 flies. SesB and ATPsyn-α were detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( C ) Purified catalytically active Dcp - 1FL , but not catalytically inactive Dcp - 1C < A , in vitro translated Drice . Cleavage of in vitro translated SesB was not detected .

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

(C) Purified catalytically active Dcp-1FL, but not catalytically inactive Dcp-1C<A, cleaved in vitro translated Drice. Cleavage of in vitro translated SesB was not detected.


( D ) V5 - tagged Dcp - 1C < A or V5 vector only control was expressed in l ( 2 ) mbn subjected to nutrient - full or starvation conditions for 2 h . After IP with anti - V5 antibodies , lysates were separated by SDS - PAGE . were visualized with colloidal Coomassie stain .

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

(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2)mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.


( E ) FLAG - SesB or a vector only control was expressed in l ( 2 ) mbn was immunoprecipitated with FLAG - agarose . Immunoblots show interaction between FLAG - SesB endogenous pro - Dcp - 1 . asterisk represents a nonspecific band .

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

(E) FLAG-SesB or a vector only control was expressed in l(2)mbn cells and was immunoprecipitated with FLAG-agarose. Immunoblots show the interaction between FLAG-SesB and endogenous pro-Dcp-1. The asterisk represents a nonspecific band.


( A ) Ovaries from fed SesBOrg flies show reduced Ref ( 2 ) P in degenerating midstage egg chamber ( arrow ) .

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

(A) Ovaries from fed SesBOrg flies show reduced Ref(2)P staining in degenerating midstage egg chamber (arrow).


( B ) Ovaries from fed w1118 SesBOrg were analyzed by Western blotting using Ref ( 2 ) P . Actin served as a loading control . Results are representative of three independent experiments ( n = 3 ) .

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(B) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref(2)P. Actin served as a loading control. Results are representative of three independent experiments (n = 3).


( C ) SesBOrg flies expressing GFP - mCherry - DrAtg8a in germline showed increased autophagosomes autolysosomes in nondegenerating ( left ) degenerating ( right ) midstage egg chambers . Insets show autophagosomes autolysosomes ( left ) autolysosomes ( right ) in SesBOrg midstage egg chambers .

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(C) SesBOrg flies expressing GFP-mCherry-DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating (left) and degenerating (right) midstage egg chambers. Insets show autophagosomes and autolysosomes (left) and autolysosomes (right) in SesBOrg midstage egg chambers.


( D ) Quantitation of percentages of autolysosomes ( autolysosomes / total autophagic structures ) in nondegenerating ovaries from fed w1118 SesBOrg flies . 10 egg chambers were manually examined per genotype . Error bars represent means ± SD . Statistical significance was determined using a two - Student ' s t ( * * * , P < 0 . 0001 ) .

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(D) Quantitation of percentages of autolysosomes (autolysosomes/total autophagic structures) in nondegenerating ovaries from fed w1118 and SesBOrg flies. 10 egg chambers were manually examined per genotype. Error bars represent the means ± SD. Statistical significance was determined using a two-tailed Student's t test (***, P < 0.0001).


( E ) Degenerating midstage egg chambers ( arrows ) from fed SesBOrg flies are TUNEL positive .

(E) Degenerating midstage egg chambers (arrows) from fed SesBOrg flies are TUNEL positive.


( F ) Degenerating midstage egg chambers from starved control SesBOrg / FM7a ; Dcp - 1Prev1 / Dcp - 1Prev1flies contained persisting nurse cellnuclei increased Ref ( 2 ) P . n = 117 ovarioles examined . Box represents zoomed image . ( G ) Degenerating midstage egg chambers from fed SesBOrg ; Dcp - 1Prev1flies contained condensed fragmented nurse cellnuclei low of Ref ( 2 ) P . Box represents zoomed image . n = 156 ovarioles examined .

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(F) Degenerating midstage egg chambers from starved control SesBOrg/FM7a;Dcp-1Prev1/Dcp-1Prev1flies contained persisting nurse cellnuclei and increased Ref(2)P. n = 117 ovarioles examined. Box represents zoomed image. (G) Degenerating midstage egg chambers from fed SesBOrg;Dcp-1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref(2)P. Box represents zoomed image. n = 156 ovarioles examined.


( B ) Immunoblotting of brown preadipocytes ( WT - 1 ) stimulated with insulin at different doses for 5 min ( n = 3 ) . molecular corresponded to I - Afadin isoform .

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(B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform.


( C ) Immunoblotting of mature brown adipocytes ( WT - 1 ) stimulated with insulin ( 100nM ) for 5 min ( n = 4 ) .

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(C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4).


( D ) Immunoblotting of immortalized human mature brown adipocytes stimulated with insulin for 5 min ( n = 3 ) .

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(D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3).


( E ) Immunoblotting of human mature adipocytes stimulated with insulin for 5 min ( n = 3 ) .

(E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3).


( F ) Immunoblotting of isolated brown adipose tissue ( BAT ) from C57BL / 6J mice injected with 5U insulin for 5 min ( n = 4 - 6 ) .

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(F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6).


( G ) of immunoblotting of isolated BAT , subcutaneous adipose tissue ( sWAT ) visceral adipose tissue ( vWAT ) from C57BL / 6J mice after fasting or re - feeding for 2 / 6 hrs ( n = 6 ) .

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(G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6).


( H ) Immunoblotting of total Afadin S1795 phosphorylation in adipose tissue depots from 12 - week old C57BL / 6 mice fed with either chow ( CD ) or high - fat diet ( HFD ) for 6 - injected with vehicle or insulin ( n = 6 ) .

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(H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6).


( I ) Immunoblotting of Afadin S1718 phosphorylation in human Stromal vascular fractions ( SVF ) from lean , obese or Type 2 Diabetic ( T2DM ) subjects stimulated or not with insulin ( n = 6 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . BAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . BAT of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . sWAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . sWAT ; of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . Liver ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . Liver of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( G ) Quantifications of phosphorylated Insulin Receptor β phosphorylated Akt across three tissues ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( H ) Quantifications of Total IRβ / tubulin in BAT , sWAT Liver ( n = 5 - 6 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( I ) Insulin - induced lipogenesis in isolated mature adipocytes from CTRL FAKO ( Mllt4 - / - ) mice ( n = 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( A ) CRISPR / nCas9 - mediated ablation of Afadin in a clonal population termed Afdn - KO .

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(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO.


( B ) Oil red O gene expression of selected adipogenic genes in WT - 1 Afdn - KO differentiated adipocytes ( n = 3 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Wild - type , Afdn - KO Afdn - KO with re - expression FLAG - tagged WT I - Afadin or a non - phosphorylable form ( S1795A ) stimulated with vehicle or insulin ( n = 4 ) for Immunoblotting .

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(C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting.


( D ) of insulin - induced Akt phosphorylation from immunoblots . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( E ) Real - time measurement ( F ) of insulin - induced fatty - acid uptake ( n = 4 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Acetylated α - tubulin in lines ( n = 3 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(C) Acetylated α-tubulin levels in cell lines (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( D ) Acetylated α - tubulin in isolated mature adipocytes from CTRL FAKO ( Mllt4 - / - ) mice ( n = 4 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(D) Acetylated α-tubulin levels in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( E ) IR abundance measured by immunoblotting following 4 hours pre - incubation with HDAC6 inhibitor ( Tubastatin A ) in differentiated adipocytes exposed to insulin ( 100nM ) stimulation for 15 min ( n = 4 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( F ) Immunolocalization of IR ( green ) HDAC6 ( red ) in Afdn - KO Afdn - KO with re - expression FLAG - tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin . Nuclei were stained with DAPI ( blue ) . Co - localization was detected by appearance of pixels where red green overlap . Scale bars : 25 µm . Representative are shown . of IR co - localized with HDAC6 ( Mandler ' s co - localization coefficient ) was quantified ( 98 - 112 from two independent experiments ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( G ) Immunoprecipitation of IR followed by immunoblotting using an anti - acetyl - Lys antibody in adipocytes lacking Afadin or re - expressing Afadin , with or without insulin stimulation ( n = 1 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( A ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( B ) composition . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (B) Body composition. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( C ) Sirius Red of representative embedded BAT , sWAT vWAT from mice on HFD of adipocyte areas ( ~ 1000 adipocytes / genotype ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( D ) IPGTT after 12 of diet AUC . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (D) IPGTT after 12 weeks of diet and AUC. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( E ) ITT after 13 of diet AUC Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (E) ITT after 13 weeks of diet and AUC Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.