Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( C , D ) VPS4B transfection ( DIV6 + 4 ) significantly reduces dendrite complexity : at 25 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 and from 87 . 5 to 100 µm p < 0 . 01 .

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 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

Primary hippocampal neurons (DIV6+4) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (C, D) VPS4B transfection (DIV6+4) significantly reduces dendrite complexity: at 25 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001 and from 87.5 to 100 µm p < 0.01.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( E , F ) Two days after shRNA transfection neurons were treated with 1 nM NRG1 - beta 1 ( NRG1 ) or vehicle and analyzed at DIV6 + 5 . shCtrl vs . shTDP : at 25 and 87 . 5 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 . shCtrl vs . shCtrl + NRG1 : from 37 . 5 to 62 . 5 µm p < 0 . 05 . shTDP vs . shTDP + NRG1 : no significant difference .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (E, F) Two days after shRNA transfection neurons were treated with 1 nM NRG1-beta 1 (NRG1) or vehicle and analyzed at DIV6+5. shCtrl vs. shTDP: at 25 and 87.5 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001. shCtrl vs. shCtrl + NRG1: from 37.5 to 62.5 µm p < 0.05. shTDP vs. shTDP+NRG1: no significant difference.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( G , H ) Transfection of the indicated combinations of shCtrl , shTDP , ErbB4 or vector control ( DIV6 + 5 ) . shCtrl + Ctrl vs . shTDP + Ctrl : from 25 to 75 µm radius p < 0 . 001 , at 87 and 112 . 5 µm p < 0 . 01 and at 100 µm p < 0 . 05 . shTDP + Ctrl vs . shTDP + ErbB4 : from 25 to 100 µm radius p < 0 . 001 , at 112 . 5 µm p < 0 . 01 . shCtrl + Ctrl vs . shTDP + ErbB4 : no significant differences .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (G, H) Transfection of the indicated combinations of shCtrl, shTDP, ErbB4 or vector control (DIV6+5). shCtrl + Ctrl vs. shTDP + Ctrl: from 25 to 75 µm radius p < 0.001, at 87 and 112.5 µm p < 0.01 and at 100 µm p < 0.05. shTDP + Ctrl vs. shTDP + ErbB4: from 25 to 100 µm radius p < 0.001, at 112.5 µm p < 0.01. shCtrl + Ctrl vs. shTDP + ErbB4: no significant differences.


Confocal z - projections of the indicated genotypes and age , labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ - specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . While γ - axons of control flies project through the entire lobe ( C is the RNAi control ; n = 12 / 12 , E ; is the tsCRISPR control ; n = 14 / 14 ) , knockdown of dpr12 by RNAi ( D ; n = 12 / 12 ) or knockout by tsCRISPR ( F ; n = 14 / 14 ) resulted in short axons . At L3 , γ - axons in dpr12 ∆ 50 - 81 homozygotus mutant animals ( K ; n = 20 / 20 ) resemble WT γ - axons ( G ; n = 20 / 20 ) . At 48hr APF , γ - axons normally re - extend to form the adult lobe ( H ; n = 12 / 12 ) . dpr12 ∆ 50 - 81 γ - axons ( L ; n = 14 / 14 ) fail to extend to the end of the lobe . This defect persists to adult ( I ; n = 11 / 11 vs . M ; n = 18 / 18 ) . Expressing a UAS - Dpr12 transgene within γ - KCs in dpr12 ∆ 50 - 81 homozygote mutant animals rescued the axon regrowth defect ( N ; n = 23 / 24 , J ; n = 14 / 14 ) . The adult γ - lobe and α / β lobes are outlined in ( C , D ) in yellow and orange , respectively , for clarity . Asterisks demarcate the distal part of the lobe . Green and white indicate mCD8 - GFP . Magenta represents FasII staining . Scale bar is 20 µm .

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

Confocal z-projections of the indicated genotypes and age, labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ-specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). While γ-axons of control flies project through the entire lobe (C is the RNAi control; n=12/12, E; is the tsCRISPR control; n=14/14), knockdown of dpr12 by RNAi (D; n=12/12) or knockout by tsCRISPR (F; n=14/14) resulted in short axons. At L3, γ-axons in dpr12∆50-81 homozygotus mutant animals (K; n=20/20) resemble WT γ-axons (G; n=20/20). At 48hr APF, γ-axons normally re-extend to form the adult lobe (H; n=12/12). dpr12∆50-81 γ-axons (L; n=14/14) fail to extend to the end of the lobe. This defect persists to adult (I; n=11/11 vs. M; n=18/18). Expressing a UAS-Dpr12 transgene within γ-KCs in dpr12∆50-81 homozygote mutant animals rescued the axon regrowth defect (N; n=23/24, J; n=14/14). The adult γ-lobe and α/β lobes are outlined in (C, D) in yellow and orange, respectively, for clarity. Asterisks demarcate the distal part of the lobe. Green and white indicate mCD8-GFP. Magenta represents FasII staining. Scale bar is 20 µm.


Confocal z - projections of MARCM neuroblast ( NB , A - F ) and single - cell ( SC , G - L ) clones labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . At L3 , NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones ( A ; n = 20 / 20 , D ; n = 15 / 15 , G ; n = 15 / 15 and J ; n = 17 / 17 ) . At 48hr APF and adult stage , WT NB ( B ; n = 15 / 15 , C ; n = 10 / 10 ) and SC ( H ; n = 16 / 16 , I ; n = 13 / 13 ) clones extend their axons to form the full adult lobe . In contrast , clones expressing dpr12 RNAi ( E ; n = 14 / 14 , F ; n = 22 / 2 , K ; n = 18 / 24 , L ; n = 19 / 27 ) fail to extend their axons to the distal part of the medial lobe ( asterisks ) . I ' and L ' are traces of multiple single - cell clones depicting each cell in a different color .

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

Confocal z-projections of MARCM neuroblast (NB, A-F) and single-cell (SC, G-L) clones labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). At L3, NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones (A; n=20/20, D; n=15/15, G; n=15/15 and J; n=17/17). At 48hr APF and adult stage, WT NB (B; n=15/15, C; n=10/10) and SC (H; n=16/16, I; n=13/13) clones extend their axons to form the full adult lobe. In contrast, clones expressing dpr12 RNAi (E; n=14/14, F; n=22/2, K; n=18/24, L; n=19/27) fail to extend their axons to the distal part of the medial lobe (asterisks). I' and L' are traces of multiple single-cell clones depicting each cell in a different color.


Confocal z - projections of brains expressing MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; A - D ) and DIP - δGFSTF ( DIP - δ - GFP ; E - H ) fusion proteins at the indicated time points . See Figures EV 1 and 4 for more details on the fusion protein structure . ( A - D ) Dpr12 - GFP is localized to the distal part of the γ - lobe at L3 ( A ; n = 10 / 10 ) , 48hr APF ( C ; n = 12 / 12 ) and the adult stage ( D ; n = 20 / 20 ) , where it colocalizes with the γ4 > γ1γ2 MBON ( γ4 ; labeled by GMR18H09 - Gal4 driving the expression of CD4 - tdT ) . At 24hr APF ( B ; n = 10 / 10 ) , Dpr12 - GFP appears diffuse . ( E - H ) DIP - δ - GFP is localized to the distal part of the γ - lobe throughout development : L3 ( E ; n = 16 / 16 ) , 24hr APF ( F ; n = 10 / 10 ) , 48hr APF ( G ; n = 12 / 12 ) and adult ( H ; n = 24 / 24 ) . At the adult stage , DIP - δ - GFP is colocalized with the γ4 > γ1γ2 MBON ( γ4 ) .

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

Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP; A-D) and DIP-δGFSTF (DIP-δ-GFP; E-H) fusion proteins at the indicated time points. See Figures EV 1 and 4 for more details on the fusion protein structure. (A-D) Dpr12-GFP is localized to the distal part of the γ-lobe at L3 (A; n=10/10), 48hr APF (C; n=12/12) and the adult stage (D; n=20/20), where it colocalizes with the γ4>γ1γ2 MBON (γ4; labeled by GMR18H09-Gal4 driving the expression of CD4-tdT). At 24hr APF (B; n=10/10), Dpr12-GFP appears diffuse. (E-H) DIP-δ-GFP is localized to the distal part of the γ-lobe throughout development: L3 (E; n=16/16), 24hr APF (F; n=10/10), 48hr APF (G; n=12/12) and adult (H; n=24/24). At the adult stage, DIP-δ-GFP is colocalized with the γ4>γ1γ2 MBON (γ4).


Confocal z - projections DIP - δ hetero - and homozygous brains in which γ - KCs were labeled by expressing membrane bound tandem tomato ( mtdT - HA ) driven by the γ specific QF2 driver R71G10 - QF2 ( γ - QF2 ) . Larval ( L3 ) γ - axons grow normally in DIP - δT2A - Gal4 heterozygotes ( A ; n = 16 / 16 ) and homozygotes ( E ; n = 24 / 24 ) . In contrast , at 48hr APF and adult , γ - axons within DIP - δT2A - Gal4 homozygotes do not enter the distal part of the lobe ( asterisks ; F ; n = 8 / 8 , G ; n = 20 / 20 ) , while they grow normally in heterozygotes ( B ; n = 12 / 12 , C ; n = 12 / 12 ) . Overexpression of a DIP - δ transgene driven by the Gal4 activity of DIP - δT2A - Gal4 ( see also Figure S4 ) does not affect normal growth ( D ; n = 10 / 10 ) and rescues mutant phenotypes ( H ; n = 20 / 20 ) . Note that while the R71G10 driver is consistently expressed in γ - KCs , it is also expressed in α / β - KCs in a stochastic manner . The adult γ - lobe and α / β lobes are outlined in C - D in yellow and orange , respectively , for clarity .

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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Confocal z-projections DIP-δ hetero- and homozygous brains in which γ-KCs were labeled by expressing membrane bound tandem tomato (mtdT-HA) driven by the γ specific QF2 driver R71G10-QF2 (γ-QF2). Larval (L3) γ-axons grow normally in DIP-δT2A-Gal4 heterozygotes (A; n=16/16) and homozygotes (E; n=24/24). In contrast, at 48hr APF and adult, γ-axons within DIP-δT2A-Gal4 homozygotes do not enter the distal part of the lobe (asterisks; F; n=8/8, G; n=20/20), while they grow normally in heterozygotes (B; n=12/12, C; n=12/12). Overexpression of a DIP-δ transgene driven by the Gal4 activity of DIP-δT2A-Gal4 (see also Figure S4) does not affect normal growth (D; n=10/10) and rescues mutant phenotypes (H; n=20/20). Note that while the R71G10 driver is consistently expressed in γ-KCs, it is also expressed in α/β-KCs in a stochastic manner. The adult γ-lobe and α/β lobes are outlined in C-D in yellow and orange, respectively, for clarity.


Confocal z - projections of brains expressing DIP - δ - RNAi driven by the indicated Gal4 . γ - KCs are labeled by mtdT - HA driven by R71G10 - QF2 ( γ - QF2 ) . Expression of DIP - δ - RNAi in all glia ( Repo - Gal4 , J ; n = 28 / 28 ) or all KCs ( OK107 - Gal4 , K ; n = 12 / 12 ) did not affect γ - neuron regrowth . In contrast , expression of DIP - δ - RNAi in all postmitotic neurons ( C155 - Gal4 , L ; n = 9 / 12 ) or DIP - δ expressing neurons ( DIP - δT2A - Gal4 , M ; n = 21 / 22 ) induced a defect in γ4 / 5 innervation by γ - axons .

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

Confocal z-projections of brains expressing DIP-δ-RNAi driven by the indicated Gal4. γ-KCs are labeled by mtdT-HA driven by R71G10-QF2 (γ-QF2). Expression of DIP-δ-RNAi in all glia (Repo-Gal4, J; n=28/28) or all KCs (OK107-Gal4, K; n=12/12) did not affect γ-neuron regrowth. In contrast, expression of DIP-δ-RNAi in all postmitotic neurons (C155-Gal4, L; n=9/12) or DIP-δ expressing neurons (DIP-δT2A-Gal4, M; n=21/22) induced a defect in γ4/5 innervation by γ-axons.


( A - D ) Confocal z - projections of brains expressing DIP - δGFSTF ( DIP - δ - GFP ) together with the indicated Gal4s and transgenes . Expressing diphtheria toxin ( DTi ) and the membrane bound RFP ( mCD8 - RFP ; CD8 ) driven by the γ4 > γ1γ2 MBON driver MB294B - Gal4 ( MBONγ4 > γ1γ2 - Gal4 ) did not affect DIP - δ - GFP expression ( A , n = 16 / 16 ; B , n = 14 / 14 ) . In contrast , similar expression of DTi and membrane bound Tomato ( CD4 - tdT ; CD4 ) in PAM - DANs ( using the GMR58E02 - Gal4 ; PAM - DAN - Gal4 ) abolished the normal DIP - δ - GFP expression in the γ4 / 5 zone ( compare D , n = 18 / 18 , to C , n = 16 / 16 ) and within the PAM - DAN cell bodies ( compare D ' to C ' ) . Magenta is CD8 - RFP ( A - B ) , CD4 - mtdT ( C - D ) . Green is GFP , grayscale depict individual channels as labeled . Scale bar is 20 µm . Yellow dashed line demarcates the γ - lobe based on FasII staining ( not shown ) .

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

(A-D) Confocal z-projections of brains expressing DIP-δGFSTF (DIP-δ-GFP) together with the indicated Gal4s and transgenes. Expressing diphtheria toxin (DTi) and the membrane bound RFP (mCD8-RFP; CD8) driven by the γ4>γ1γ2 MBON driver MB294B-Gal4 (MBONγ4>γ1γ2-Gal4) did not affect DIP-δ-GFP expression (A, n=16/16; B, n=14/14). In contrast, similar expression of DTi and membrane bound Tomato (CD4-tdT; CD4) in PAM-DANs (using the GMR58E02-Gal4; PAM-DAN-Gal4) abolished the normal DIP-δ-GFP expression in the γ4/5 zone (compare D, n=18/18, to C, n=16/16) and within the PAM-DAN cell bodies (compare D' to C'). Magenta is CD8-RFP (A-B), CD4-mtdT (C-D). Green is GFP, grayscale depict individual channels as labeled. Scale bar is 20 µm. Yellow dashed line demarcates the γ-lobe based on FasII staining (not shown).


( E - G ) Confocal z - projections of MARCM clones labeled by DIP - δT2A - Gal4 ( DIP - δ Gal4 ) driving the expression of membrane bound GFP ( mCD8 - GFP ; CD8 ) and heat shocked at 24hr after egg laying . Clones innervate the γ4 / 5 zones at 24hr APF ( E ; n = 8 ) , 48hr APF ( F ; n = 8 ) and adult ( G ; n = 16 ) . Clones become tyrosine hydroxylase ( TH ) positive only at 48hr APF onwards ( F , G ) . Magenta is FasII , green is mCD8 - GFP , cyan is TH antibody staining , grayscale single channels are shown as indicated . White dashed line demarcates the γ - lobe . Scale bar is 20 µm .

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

(E-G) Confocal z-projections of MARCM clones labeled by DIP-δT2A-Gal4 (DIP-δ Gal4) driving the expression of membrane bound GFP (mCD8-GFP; CD8) and heat shocked at 24hr after egg laying. Clones innervate the γ4/5 zones at 24hr APF (E; n=8), 48hr APF (F; n=8) and adult (G; n=16). Clones become tyrosine hydroxylase (TH) positive only at 48hr APF onwards (F,G). Magenta is FasII, green is mCD8-GFP, cyan is TH antibody staining, grayscale single channels are shown as indicated. White dashed line demarcates the γ-lobe. Scale bar is 20 µm.


( A - J ) Confocal z - projections of brains expressing MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ) and DIP - δGFSTF ( DIP - δ - GFP ) fusion proteins of the indicated genotypes and time points . ( A - D ) Dpr12 - GFP expression is diffuse in DIP - δT2A - Gal4 homozygotes mutant brains at L3 ( A ; n = 20 / 20 ) , 24hr APF ( B ; n = 14 / 14 ) , 48hr APF ( C ; n = 28 / 28 ) and adult ( D ; n = 26 / 26 ) . ( E - H ) DIP - δ - GFP expression in dpr12 ∆ 50 - 81 homozygotes mutant brains remains localized to the distal part of the γ - lobe at L3 ( E ; n = 16 / 16 ) , 24hr APF ( F ; n = 16 / 16 ) , and 48hr APF ( G ; n = 10 / 10 ) but cannot be identified in adult brains ( H ; n = 16 / 16 ) . ( I - J ) Dpr12 - GFP expression in WT animals ( I , n = 8 / 8 ) or in those ectopically expressing DIP - δ in PAM - DANs that innervate the γ3 zone ( J , n = 14 / 14 ) driven by MB441B - Gal4 ( PAM - DAN - γ3 - Gal4 ) . DIP - δ expression in PAM - DAN - γ3 resulted in Dpr12 - GFP localization within the γ3 zone ( arrow ) , in addition to its normal γ4 / γ5 localization .

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(A-J) Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP) and DIP-δGFSTF (DIP-δ-GFP) fusion proteins of the indicated genotypes and time points. (A-D) Dpr12-GFP expression is diffuse in DIP-δT2A-Gal4 homozygotes mutant brains at L3 (A; n=20/20), 24hr APF (B; n=14/14), 48hr APF (C; n=28/28) and adult (D; n=26/26). (E-H) DIP-δ-GFP expression in dpr12∆50-81 homozygotes mutant brains remains localized to the distal part of the γ-lobe at L3 (E; n=16/16), 24hr APF (F; n=16/16), and 48hr APF (G; n=10/10) but cannot be identified in adult brains (H; n=16/16). (I-J) Dpr12-GFP expression in WT animals (I, n=8/8) or in those ectopically expressing DIP-δ in PAM-DANs that innervate the γ3 zone (J, n=14/14) driven by MB441B-Gal4 (PAM-DAN-γ3-Gal4). DIP-δ expression in PAM-DAN-γ3 resulted in Dpr12-GFP localization within the γ3 zone (arrow), in addition to its normal γ4/γ5 localization.


Confocal z - projections of dpr12 ∆ 50 - 81 heterozygous ( A , n = 15 ; C , n = 10 ; E , n = 10 ) and homozygous brains ( B , n = 8 ; D , n = 18 ; F , n = 24 ) expressing mCD8 - GFP ( CD8 ) driven by ( E , F ) R18H09 - Gal4 ( MBONγ4 > γ1γ2 - Gal4 ) . The greyscale channels are sub - z - projections comprised of slices restricted to the γ - lobe region . White dashed line demarcates the γ - lobe . Bottom : Cartoons schematizing MB lobe structure and innervation by specific PAM - DANs or MBON .

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Confocal z-projections of dpr12∆50-81 heterozygous (A, n=15; C, n=10; E, n=10) and homozygous brains (B, n=8; D, n=18; F, n=24) expressing mCD8-GFP (CD8) driven by (E, F) R18H09-Gal4 (MBONγ4>γ1γ2 -Gal4). The greyscale channels are sub-z-projections comprised of slices restricted to the γ-lobe region. White dashed line demarcates the γ-lobe. Bottom: Cartoons schematizing MB lobe structure and innervation by specific PAM-DANs or MBON.


Single confocal slices of WT ( G , n = 5 / 5 ) and dpr12 ∆ 50 - 81 homozygous brains ( H , n = 5 / 5 ) stained with anti - Brp . Dashed lines demarcate neuropil boundaries , schematic shown below . Cre , crepine , a neuropil that surrounds the medial MB lobes ; AL , antenna lobe .

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Single confocal slices of WT (G, n=5/5) and dpr12∆50-81 homozygous brains (H, n=5/5) stained with anti-Brp. Dashed lines demarcate neuropil boundaries, schematic shown below. Cre, crepine, a neuropil that surrounds the medial MB lobes; AL, antenna lobe.


( C , D ) Confocal z - projections of adult DIP - δT2A - Gal4 / 1 - 119 trans - heterozygous mutant ( D ) or DIP - δ1 - 119 / + heterozygous ( C ) brains , which express MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; green ) , in which DIP - δ - Gal4 either drives expression or UAS - DIP - α ( D ) or not ( C ) . Grayscale panels represent single channels , as indicated . The γ - lobe is outlined in white .

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(C, D) Confocal z-projections of adult DIP-δT2A-Gal4/1-119 trans-heterozygous mutant (D) or DIP-δ1-119/+ heterozygous (C) brains, which express MiMIC mediated Dpr12GFSTF (Dpr12-GFP; green), in which DIP-δ-Gal4 either drives expression or UAS-DIP-α (D) or not (C). Grayscale panels represent single channels, as indicated. The γ-lobe is outlined in white.


( E , F ) Confocal z - projections of adult dpr12Δ50 - 81 homozygous mutant brains , in which DIP - δ - Gal4 drives expression of either UAS - mtdT ( E ) or UAS - DIP - α - T2A - tdT ( F ; expected to induce expression of DIP - α as well as tdT encoded by a polycistronic message ) . In orange are longitudinal sections across the γ - lobe at the indicated location . The γ - lobe is outlined in white , as determined by FasII staining ( grey in E ' - F ' ) . Cyan is tdT within DIP - δ + cells . Grey boxes below the images describe the relevant components within PAM - DANs and γ - KCs .

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(E, F) Confocal z-projections of adult dpr12Δ50-81 homozygous mutant brains, in which DIP-δ-Gal4 drives expression of either UAS-mtdT (E) or UAS-DIP-α-T2A-tdT (F; expected to induce expression of DIP-α as well as tdT encoded by a polycistronic message). In orange are longitudinal sections across the γ-lobe at the indicated location. The γ-lobe is outlined in white, as determined by FasII staining (grey in E'-F'). Cyan is tdT within DIP-δ+ cells. Grey boxes below the images describe the relevant components within PAM-DANs and γ-KCs.


A . Stills depicting actin flow in extracellular RH , myoA KO and adf KD parasites expressing Cb EmeraldFP . Continuous F - actin flow can be seen from the apical tip , to the Golgi region towards the posterior pole of the parasite . No difference can be observed between RH and myoA KO . For adf KD , the flow is completely abrogated . Red arrowhead marks the Golgi region , while the yellow arrowhead marks the apical nucleation centre . B . Skeletonisation processing of ( A ) depicting areas where F - actin dynamics / flow is prevalent . Individual signals for F - actin form a continuous , dynamic network that connects apical and posterior pole of the parasite . Red arrowhead marks the Golgi region nucleation centre , while the yellow arrowhead marks the apical nucleation centre . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin . C . Kymograph analysis of ( A ) and ( B ) . The kymograph was generated by tracing a line in the middle of the parasite ' s body ( red line ) . The colour - coded kymograph represents forward movement ( red ) , backwards movement ( green ) and static F - actin ( blue ) . The results demonstrate continuous exchange of F - actin from the apical to the posterior pole of the parasite within the cytosol of the parasite . This cytosolic exchange is similar in RH and myoA KO parasites , while completely abolished in the case of adf KD . Data information : Numbers indicate minutes : seconds ; and Scale bars represent 5 µm .

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A. Stills depicting actin flow in extracellular RH, myoA KO and adf KD parasites expressing Cb EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. B. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. C. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backwards movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD. Data information: Numbers indicate minutes:seconds; and Scale bars represent 5 µm.


D . Top three rows : Parasites expressing Cb EmeraldFP along with myoA - SNAP before and after Ca2 + - Ionophore ( A23187 ) or BIPPO treatment . The parasites were also treated with 2 µM of Cytochalasin D or DMSO ( as control ) for 30 min . After addition of A23187 , preferential relocalisation of actin can be observed at the apical tip , while addition of BIPPO caused F - actin accumulation at the basal end . 4th row : myoAKO parasites expressing Cb - EmeraldFP before and after treatment with A23187 and BIPPO . While no apical or basal accumulation of F - actin is observed , some peripheral location of F - actin occurs in presence of A23187 or BIPPO , preferentially in the apical half of the parasite . Bottom row : adf cKD parasites expressing Cb - EmeraldFP before and after treatment with Ca2 + Ionophore or BIPPO . No apparent change in F - actin localisation can be seen upon treatment . E . Skeletonization of movies shown in D . Before addition of A23187 or BIPPO , RH and myoA KO parasites behave similar with no accumulation of F - actin at the apical tip . After treatment with calcium ionophore A23187 , preferential accumulation of F - actin at the apical tip can be observed for RH ( red arrowhead ) , while BIPPO presented preferential accumulation in the basal end ( red arrowhead ) . In myoA KO , some relocalisation of actin is observed at the periphery of the parasite ( yellow arrowhead ) . In the case of adf cKD , no relocalisation occurs . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin .

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D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.


F . Quantification of actin accumulation before and after treatment with Ca2 + Ionophore or BIPPO . The parasites were counted for F - actin accumulation after adding Ca2 + Ionophore or BIPPO . Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip . A minimum of 200 parasites were counted upon 3 biological replicates . Two - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . Error bars represent standard deviation .

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F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.


A . Wild Type RH parasites mid - invasion . Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes , fixed with 4 % PFA before labelling the TJ with anti - Ron2 ( yellow ) and DAPI ( magenta ) for nuclear staining . White arrow points at invasion direction .

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A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.


B . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . 100 parasites found mid - invasion were counted in triplicate and compared to extracellular , freshly egressed parasites . One - way ANOVA was used for statistical analysis . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . and Scale bars represent 5 µm .

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B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001. Data information: Error bars represent standard deviation. and Scale bars represent 5 µm.


C . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . Indicated parasites were analysed . For adf cKD induced and non - induced conditions were compared , as indicated . 30 parasites were counted for each condition in triplicate . One - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . * * * p value 0 . 0009 . Data information : Error bars represent standard deviation . ; and Scale bars represent 5 µm .

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C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009. Data information: Error bars represent standard deviation. ; and Scale bars represent 5 µm.


D . Time - lapse microscopy analysis of indicated parasites invading HFFs cells . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes :

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D. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


E . Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations . Individual stages were named in regard to the invasion step ( Penetration initiation , Speed decrease and finalisation ) . Invasion was analysed with Icy Image Processing Software ( Pasteur Institut ) using the Manual Tracking plugin by analysing each movie 10 times and averaging the

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E. Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurements


F . Time - lapse analysis demonstrating abortive invasion of indicated parasites . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes :

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F. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


A . Time - lapse analysis depicting invading RH adfcKD parasites expressing Cb - EmeraldFP . A F - actin ring can be observed at the TJ in the majority of cases ( red arrowheads , first panel ) . Invasion can also occur without formation of an F - actin ring ( second panel ) . During capped invasion , an F - actin ring is formed ( third panel ) . Invasion of adf cKD parasites occurs without formation of an F - actin ring ( fourth panel ) . F - actin accumulation is observed at the posterior pole of the parasite in all cases ( orange arrows ) . Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in ( B ) . Yellow and purple dotted lines indicate the extra - and intracellular part of the parasite respectively . B . Intensity plot profiles of the movies shown in ( A ) . Depicted plots correlate to half - invaded parasites . In the case of F - actin ring formation , two distinct peaks are detected , correlating to the F - actin ring at the TJ ( red arrowhead ) and the posterior pole of the parasite ( orange arrow ) . In the case of no F - actin ring , no accumulation at the TJ can be observed ; only the peak correlating to posterior F - actin can be detected . For adf cKD , only one peak can be seen corresponding to the posterior pole of the parasite . C . Quantification of F - actin accumulation at the TJ for RH Cb - EmeraldFP parasites as detected in time lapse analysis . The values are expressed as percentage . 28 total parasite invasion events were captured across three different biological replicates . Two - tailed unpaired t - test analysis was performed . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm . * * * * p value < 0 . 0001 .

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A. Time-lapse analysis depicting invading RH adfcKD parasites expressing Cb-EmeraldFP. A F-actin ring can be observed at the TJ in the majority of cases (red arrowheads, first panel). Invasion can also occur without formation of an F-actin ring (second panel). During capped invasion, an F-actin ring is formed (third panel). Invasion of adf cKD parasites occurs without formation of an F-actin ring (fourth panel). F-actin accumulation is observed at the posterior pole of the parasite in all cases (orange arrows). Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in (B). Yellow and purple dotted lines indicate the extra- and intracellular part of the parasite respectively. B. Intensity plot profiles of the movies shown in (A). Depicted plots correlate to half-invaded parasites. In the case of F-actin ring formation, two distinct peaks are detected, correlating to the F-actin ring at the TJ (red arrowhead) and the posterior pole of the parasite (orange arrow). In the case of no F-actin ring, no accumulation at the TJ can be observed; only the peak correlating to posterior F-actin can be detected. For adf cKD, only one peak can be seen corresponding to the posterior pole of the parasite. C. Quantification of F-actin accumulation at the TJ for RH Cb-EmeraldFP parasites as detected in time lapse analysis. The values are expressed as percentage. 28 total parasite invasion events were captured across three different biological replicates. Two-tailed unpaired t-test analysis was performed. **** p value <0.0001. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm. ****p value <0.0001.


D . Representative immunofluorescence assays on indicated parasite strains during host cell penetration . Parasites were labelled with indicated antibodies . SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite . Antibodies against Ron2 were used to stain the TJ . Red arrowhead : position of the TJ . Orange arrow : posterior pole of the parasite . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm .

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D. Representative immunofluorescence assays on indicated parasite strains during host cell penetration. Parasites were labelled with indicated antibodies. SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite. Antibodies against Ron2 were used to stain the TJ. Red arrowhead: position of the TJ. Orange arrow: posterior pole of the parasite. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm.


E . Tight Junction assay on fixed samples depicting invasion rates of indicated parasites . While in the majority of control parasites F - actin can be detected at the junction ( ~ 80 % ) , no junctional ring can be detected in ~ 20 % of all invasion events . In contrast , adf cKD , act1 cKO or myoA KO only show an overall invasion rate of ~ 20 % compared to controls . In these cases junctional F - actin accumulation could never be detected . Note that the number of total invasion events for adf cKD , act1cKO and myoAKO correlates to the number of invasion events , seen for RH without F - actin ring formation . Data information : Error bars represent standard deviation . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 .

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E. Tight Junction assay on fixed samples depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation. Data information: Error bars represent standard deviation. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


F . Quantification of posterior F - actin accumulation during invasion of indicated parasite lines . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 .

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F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


A . Time - lapse analysis of invading RH Cb - EmeraldFP parasites . During penetration , an F - actin ring is formed at the TJ ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) and posterior F - actin appears to be directly connected to the nucleus and TJ ( blue arrow ) . B . Analysis of nucleus deformation during live imaging shown in A . The nucleus constricts while its passing through the TJ ( red arrowheads ) . C . The posterior end of the parasite deforms during the invasion process . F - actin is accumulated during invasion , with the posterior end contracting ( blue arrow ) . Data information : Numbers indicate minutes : seconds . Scale bar represents 5 µm .

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A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads). C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow). Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm.


D . Time - lapse stills depicting RH Cb - EmeraldFP parasites invading in absence of detectable F - actin at the junction ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) during invasion . Actin accumulation at the posterior pole of the parasite is strongly detected in all cases ( blue arrow ) . E . Analysis of nucleus deformation during live imaging shown in ( D ) . The nucleus appears to not show constriction once its passing through the TJ when the F - actin ring is not present . F . The posterior end of the parasite also deforms during the invasion process .

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D. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present. F. The posterior end of the parasite also deforms during the invasion process.


G . Z - stack gallery depicting mid - invading myoA KO parasites . Note that the nucleus is located located at the back during invasion events . Data information : Scale bar represents 5 µm . White arrow points to direction of invasion .

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G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events. Data information: Scale bar represents 5 µm. White arrow points to direction of invasion.


A . SR - SIM images depicting invading parasites with and without the presence of the F - actin ring at the tight junction and invading adf cKD Cb - EmeraldFP parasites . SAG1 staining ( in red ) was performed prior to permeabilisation to specifically label extracellular part of the parasite . Note the accumulation of actin ( blue arrow ) around the nucleus and the posterior pole of the parasite , irrespective of F - actin ring formation at the TJ ( orange arrowhead ) . 3D rendering is shown for each presented case ( inlet ) . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


B . SR image of an invading parasite before nuclear entry . F - actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole . C . F - actin localisation in relation to the TJ during invasion . F - actin ( white arrow ) is in close vicinity to the Ron2 ring ( light blue arrow ) during invasion . Data information : cale bar represents 5 µm for SR - SIM images hite arrow points to direction of invasion .

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B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion.


D . Quantification of perinuclear actin in indicated parasites . Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus . Still images to the right represent what was considered as F - actin - nucleus association . The orange arrowhead points to F - actin surrounding the nucleus in the posterior end of the parasite , the red and yellow arrowhead shows F - actin in the vicinity of the nucleus , hinting at a close interaction between the two . Three biological replicates were analysed with a minimum of 25 parasites per replicate . Data information : Error bars represent standard deviation . Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


E - G . SR - SIM images showing three stages of invading wt parasites . SiR - tubulin staining for microtubules ( in red ) was performed prior to fixation to specifically label microtubules in the parasite . During invasion , the microtubules ( MT ) are deformed at the TJ area ( white arrowhead ) . Accumulation of actin ( green arrow ) at the posterior forms a F - actin meshwork . The nucleus is deformed when it enters the TJ area ( orange arrowhead ) . H - J . In the case of the myoA KO strain , the MTs also deform when passing through the TJ ( white arrowhead ) . However , F - actin is not concentrated at the posterior pole or the TJ , but evenly distributed within the cytosol of the parasite , with some peripheral location . K - M . In case of adfKD , MTs are deformed at the TJ area ( white arrowhead ) . F - actin is accumulated at both ends of the parasite ( green arrowheads ) , with no accumulation at the TJ . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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

E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


A . Different invasion stages of P . falciparum merozoites expressing Cb - EmeraldFP . DAPI labels the nucleus ( DNA ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and anti - RON4 labels the junction ( red ) . Left panel : onset of invasion . Middle panel : A merozoite in the middle of invasion ; note the constriction of the nucleus ( blue ) as it passes through the RON4 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing invasion ; note the F - actin ring beyond the RON4 - junction and actin filaments surrounding the nucleus . Data information : Scale bar represents 1 µm for Plasmodium

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A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparum


B . IFA showing various stages of invasion of erythrocytes by P . knowlesi merozoites . DAPI labels the nucleus ( DNA ) , anti - actin Antibody labels actin filaments ( anti - actin , green ) and the invasion junction is marked with a C - terminally tagged PkRON2 mCherryHA ( red ) . Left panel : a merozoite beginning invasion ; note the formation of an F - actin ring ( green ) behind the PkRON2 junction ( red ) . Middle panel : A merozoite in the middle of the invasion event ; note the constriction of the nucleus ( blue ) as it passes through the PkRON2 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing the process of invasion ; note the F - actin ring beyond the PkRON2 - junction and actin filaments colocalising with the nucleus . Data information : Scale bar represents 3 µm for Plasmodium knowlesi .

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B. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.


C . Super - resolution images showing various stages of invasion of erythrocytes by P . falciparum merozoites expressing Cb - EmeraldFP . Left panel : DAPI labels the nucleus ( blue ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and the invasion junction is marked with an anti - RON4 antibody ( red ) . Brightfield images have been marked with red dots depicting the junction , black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell . Top panel shows an attached merozoite beginning the process of invasion , the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion . Right panel : Schematic depicting merozoite invasion into red blood cells . Letters depict the corresponding event in the model ( left ) and microscopy pictures ( right ) . White arrows depict actin filaments that colocalise with the nucleus . D . Quantification of Plasmodium falciparum invasion events categorising actin - TJ and actin - nucleus association events . 121 events were counted in total . Data information : Error bars represent standard deviation . Scale bar represents 1 µm for Plasmodium

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C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum


( A ) MYB knockdown ( MiaPaCa - shMYB and Panc1 - shMYB ) and forced MYB - overexpressing ( BxPC3 - MYB ) PC cells along with their respective control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were subjected to hypoxia ( 1 % O2 ) treatment for different time intervals ( 0 - 96 h ) . Their growth was measured by viable cell counting using a trypan blue exclusion assay .

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(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.


( E , F ) ECAR ( E ) and OCR ( F ) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 ( 100 μM for 4 h ) . Thereafter , ECAR was measured in PC cells with sequential injection of glucose , oligomycin , and 2 - DG , while OCR was measured with the serial addition of oligomycin , FCCP , and antimycin A / rotenone .

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(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.


( A ) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals . HIF1α accumulation was examined as a positive control .

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(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.


MYB protein ( C ) levels were measured in the prostate ( LNCaP ) , lung ( A549 ) , breast ( MB231 ) , and colon ( HCT116 ) cancer cells cultured under normoxia and hypoxia ( 6 h ) by immunoblotting respectively . β - actin served as an internal control . N - normoxia and H - hypoxia .

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MYB protein (C) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting respectively. β-actin served as an internal control. N- normoxia and H- hypoxia.


( E ) Pancreatic cancer cells were transiently transfected either with non - targeted control siRNA ( siCtrl ) or HIF1α - targeting siRNA ( siHIF1α ) for 24 h and then incubated under hypoxia for 6 h . MYB expression was analyzed at protein and mRNA levels .

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(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.


( F ) Cells were treated with either vehicle or CoCl2 ( 100µM ) for 6 h , and total protein and RNA were isolated . The expression of HIF1α and MYB was examined . β - actin was used as an internal control .

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(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.


( B ) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h . HuR ( green ) , and nucleus ( blue ) . Scale bar , 25 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles .

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(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.


( C ) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting . α - Tubulin ( nuclear ) and Lamin A ( cytoplasm ) were used as loading controls .

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(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.


( G ) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h , and MYB and HuR expression was determined by immunoblotting or qRT - PCR .

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(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.


( A ) MYB - silenced ( MiaPaCa - shMYB and Panc1 - shMYB ) and MYB - overexpressing ( BxPC3 - MYB ) PC cells , along with their control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were exposed to hypoxia for 6 h . Protein lysates were made , and the expression of MYB and HIF1α was examined by immunoblotting . β - actin served as a loading control .

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(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.


( C ) The transcriptional activity of the HIF1α promoter was determined by performing the promoter - reporter assay . MYB - modulated PC cell lines were transfected with the reporter plasmid , and after 24 h of transfection , cells were cultured under hypoxia or normoxia for 24 h , and the supernatant was collected . HIF1α promoter - driven Gaussia luciferase activity was normalized with SEAP activity , and the data was presented as a bar diagram .

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(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.


( E ) MYB - silenced or - overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti - MYB antibodies or IgG control followed by qPCR analysis using site - specific primers .

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(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.


( A ) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB - silenced ( shMYB ) and those having a forced overexpression of HIF1α ( shMYB - HIF1αOE ) along with their respective controls ( Scr and shMYB - Neo ) cultured under hypoxia . β - actin served as a loading control .

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(A) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB-silenced (shMYB) and those having a forced overexpression of HIF1α (shMYB-HIF1αOE) along with their respective controls (Scr and shMYB-Neo) cultured under hypoxia. β-actin served as a loading control.


( C ) Live / Dead staining was performed in MYB - silenced MiaPaCa and Panc - 1 or MYB silenced - HIF1α overexpressed cells cultured under hypoxia for 48 h . Cells were visualized under a fluorescence microscope . Representative fluorescence micrographs are presented . The number of live and dead cells was counted from the captured random images . Scale bar , 150 μm ( lower right corner ) .

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(C) Live/Dead staining was performed in MYB-silenced MiaPaCa and Panc-1 or MYB silenced- HIF1α overexpressed cells cultured under hypoxia for 48 h. Cells were visualized under a fluorescence microscope. Representative fluorescence micrographs are presented. The number of live and dead cells was counted from the captured random images. Scale bar, 150 μm (lower right corner).


( F ) MYB - silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h . Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels . The data was normalized with the number of cells and presented as relative fold changes .

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(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.


( C ) Quantitative ChIP - qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes ( GLUT3 , HK2 , PFKL , ENO2 , and MCT4 ) using anti - MYB or IgG control antibodies . The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia .

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(C) Quantitative ChIP-qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes (GLUT3, HK2, PFKL, ENO2, and MCT4) using anti-MYB or IgG control antibodies. The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia.


p300 ( E ) , and H3K9ac ( F ) on the promoter regions were also confirmed by ChIP - qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG .

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p300 (E), and H3K9ac (F) on the promoter regions were also confirmed by ChIP- qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG.


( A , B ) Interaction of MYB with HIF1α ( A ) and p300 ( B ) was examined by co - immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h . Immunoprecipitation with IgG isotype antibody was used as a control . 10 % of total cell lysate was used as input for western blot analysis .

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(A, B) Interaction of MYB with HIF1α (A) and p300 (B) was examined by co-immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h. Immunoprecipitation with IgG isotype antibody was used as a control. 10% of total cell lysate was used as input for western blot analysis.


( C , D ) Co - localization of MYB with HIF1α ( C ) and p300 ( D ) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy . Scale bar , 30 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles . For ease of visualization , a pseudo color ( purple ) was adopted for p300 staining .

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(C, D) Co-localization of MYB with HIF1α (C) and p300 (D) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy. Scale bar, 30 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles. For ease of visualization, a pseudo color (purple) was adopted for p300 staining.


( B , C ) Tumor growth was monitored by non - invasive bioluminescence imaging . Tumors were resected at the experimental endpoint , and their weights ( B ) and volumes ( C ) were recorded .

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(B, C) Tumor growth was monitored by non-invasive bioluminescence imaging. Tumors were resected at the experimental endpoint, and their weights (B) and volumes (C) were recorded.


( G ) Tumor xenograft serial sections were immunostained for pimonidazole ( PDZ ) adduct , MYB , HK2 , GLUT3 , and MCT4 using specific antibodies . Images were taken at 40X magnification . Scale bar , 60 μm ( lower left corner ) . The dashed lines demarcate the hypoxic region based on the PDZ staining pattern .

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(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.


( A - C ) AFM and TEM analysis of unloaded ( u - NPs ) and cholesterol loaded NPs ( NPs - Chol ) . AFM " height " images ( left column ) , 3D reconstruction ( middle column ) and TEM micrograph ( right column ) of u - NPs ( A ) , NPs - Chol1 ( B ) and NPs - Chol3 ( C ) .

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(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C).


( D ) Release profile in water of cholesterol ( continuous line , - ) and NBD - Chol ( dotted line , - - - ) from NPs - Chol1 and NPs - NBD - Chol1 , respectively .

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(D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively.


( E ) In vitro release of NBD - Chol from NPs at different time - intervals in NS cells . Data in the graph represent mean ( µg ) ± SEM of total NBD - Chol ( embedded into and released from NPs ; red columns ) and NBD - Chol released after NPs degradation ( purple columns ) present in the homogenates of NS cells treated with NPs - NBD -

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(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1


( A - B ) Representative confocal images of liver ( A ) and brain ( B ) slices from R6 / 2 mice ip injected with C - NPs ( left ) or with g7 - NPs ( right ) and sacrificed after 4 hrs . ( C ) Quantification of g7 - NPs localized in the liver , striatum , and cortex of WT ( n = 3 ) and R6 / 2 mice ( n = 3 ) . Data represent mean ( µg ) ± SEM .

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(A-B) Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 hrs. (C) Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n=3) and R6/2 mice (n=3). Data represent mean (µg) ± SEM.


( D - E ) g7 - NPs in brain slices from R6 / 2 mice administered with a single ip injection and sacrificed after 24 hrs ( D , left ) or 2 weeks ( D , right ) and after multiple ip injections within 1 week ( E ) .

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(D-E) g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 hrs (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E).


( F - I ) Representative confocal images of immunostaining against IBA1 ( F ) , GFAP ( G ) , calbindin ( H ) , and DARPP - 32 ( I ) on coronal sections of brains isolated from R6 / 2 mice ip injected with g7 - NPs and sacrificed at the indicated time points . White arrowheads indicate intracellular g7 - NPs .

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(F-I) Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs.


( A ) Representative confocal image ( crop ) of brain slices from R6 / 2 mice ip injected with rhodamine labeled g7 - NPs - NBD - Chol and sacrificed after 12 hrs , showing co - localization of NBD - Chol ( green ) and Rhodamine ( NPs , red ) . Scale bar : 5m .

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(A) Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine labeled g7-NPs-NBD-Chol and sacrificed after 12 hrs, showing co-localization of NBD-Chol (green) and Rhodamine (NPs, red). Scale bar: 5m.


( B - C ) Representative confocal image ( low magnification ) of brain slices from R6 / 2 mice ip injected with g7 - NPs - NBD - Chol and sacrificed after 24 hrs ( B ) or 2 weeks ( C ) and relative co - localization of NBD - Chol and g7 - NPs . Scale bar : 10 m ( D ) g7 - NPs quantification in brain slices at the same time points in ( B - C ) . DAPI was used to counterstain nuclei . Data in ( D ) are expressed as number of g7 - NPs ( evaluated based their size ) for 100 m2 sem . Statistics : * * P < 0 , 01 ( 48 hrs vs 7 days ; 7 days vs 14 days ) , * * * P < 0 , 001 ( 24 hrs vs 7 days ; 7 days vs 14 days ) determined by one - way ANOVA followed by Newmann - Keuls multiple comparison test .

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(B-C) Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 hrs (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 m (D) g7-NPs quantification in brain slices at the same time points in (B-C). DAPI was used to counterstain nuclei. Data in (D) are expressed as number of g7-NPs (evaluated based their size) for 100 m2 sem. Statistics: **P<0,01 (48 hrs vs 7 days; 7 days vs 14 days), ***P<0,001 (24 hrs vs 7 days; 7 days vs 14 days) determined by one-way ANOVA followed by Newmann-Keuls multiple comparison test.


( A , D ) Spontaneous IPSCs ( A ) and spontaneous EPSCs ( D ) were recorded from striatal MSNs ( WTs = 52 ; R6 / 2 - untreated = 27 ; R6 / 2 - Chol = 29 ) at a holding potential of + 10 mV and - 70mV respectively . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled . ( B , E ) Amplitude - frequency histogram and average frequency ( inset ) of IPSCs ( B ) and EPSCs ( E ) from R6 / 2 - Chol , R6 / 2 - untreated and WT MSNs . ( C , F ) Cumulative inter - event histogram showing the release probability of IPSCs ( C ) and EPSCs ( F ) in all groups .

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(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.


( B ) Fall latency from an accelerating rotarod for 5 - 11 - week - old WT and R6 / 2 mice during cholesterol supplementation ( WT = 17 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 13 ) .

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(B) Fall latency from an accelerating rotarod for 5-11-week-old WT and R6/2 mice during cholesterol supplementation (WT=17; R6/2-untreated=21; R6/2-Chol=13).


( C ) Rearing activity in open field at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(C) Rearing activity in open field at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( D ) global activity in the open field test at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(D) global activity in the open field test at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( E ) Index of discrimination ( % ) in WT , R6 / 2 - untreated and R6 / 2 - Chol mice during disease progression , at 8 weeks of age ( WT = 24 ; R6 / 2 - untreated = 36 ; R6 / 2 - Chol = 21 ) , at 10 weeks of age ( WT = 25 ; R6 / 2 - untreated = 35 ; R6 / 2 - Chol = 20 ) and at 12 weeks of age ( WT = 24 ; R6 / 2 - untreated = 30 ; R6 / 2 - Chol = 19 ) ; the index above zero indicates a preference for the novel object ; the index below zero indicates a preference for the familiar object . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or treated with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled .

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(E) Index of discrimination (%) in WT, R6/2-untreated and R6/2-Chol mice during disease progression, at 8 weeks of age (WT=24; R6/2-untreated=36; R6/2-Chol=21), at 10 weeks of age (WT=25; R6/2-untreated=35; R6/2-Chol=20) and at 12 weeks of age (WT=24; R6/2-untreated=30; R6/2-Chol=19); the index above zero indicates a preference for the novel object; the index below zero indicates a preference for the familiar object. As no differences were found between R6/2 mice treated with saline (R6/2) or treated with empty g7-NPs (R6/2-emp), data were pooled.


( A - B ) protein levels ( A ) and relative densitometry quantification ( B ) of several synaptic proteins in triton - insoluble ( synaptic enriched ) fractions purified from total brains . Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6 / 2 mice by cholesterol supplementation .

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(A-B) protein levels (A) and relative densitometry quantification (B) of several synaptic proteins in triton-insoluble (synaptic enriched) fractions purified from total brains. Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6/2 mice by cholesterol supplementation.


( C - D - E ) mRNA levels for Bdnf ( C ) , Snap25 ( D ) in cortex and hippocampus ; Complexin II ( E ) in cortex , hippocampus and striatum from a subset of WT ( n = 4 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - chol animals ( n = 3 ) . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D-E) mRNA levels for Bdnf (C), Snap25 (D) in cortex and hippocampus; Complexin II (E) in cortex, hippocampus and striatum from a subset of WT (n=4), R6/2-untreated (n=7) and R6/2-chol animals (n=3). As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( F - G ) Representative images of Nissl Staining ( F ) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT ( n = 7 ) , R6 / 2 ( n = 7 ) , R6 / 2 - emp ( n = 6 ) , R6 / 2 - Chol ( n = 8 )

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(F-G) Representative images of Nissl Staining (F) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT (n= 7), R6/2 (n=7), R6/2-emp (n=6), R6/2-Chol (n=8)


( A - B ) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT ( n = 6 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - Chol ( n = 5 ) mice . Data represent mean ± SEM .

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(A-B) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT (n=6), R6/2-untreated (n=7) and R6/2-Chol (n=5) mice. Data represent mean ± SEM.


( C - D ) mRNA levels of hmgcr and fdft1 in liver and lung of WT ( n = 7 ) , R6 / 2 - untreated ( n = 8 ) and R6 / 2 - Chol ( n = 4 ) mice . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D) mRNA levels of hmgcr and fdft1 in liver and lung of WT (n=7), R6/2-untreated (n=8) and R6/2-Chol (n=4) mice. As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( E - F ) mRNA levels of inflammatory genes in liver and lung of the of WT ( n = 7 ) , R6 / 2 ( n = 4 ) , R6 / 2 - emp ( n = 4 ) and R6 / 2 - Chol ( n = 5 ) mice .

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(E-F) mRNA levels of inflammatory genes in liver and lung of the of WT (n=7), R6/2 (n=4), R6/2-emp (n=4) and R6/2-Chol (n=5) mice.


A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag - USP19 ( top ) or endogenous USP19 in HeLa cells ( bottom ) treated with USP19 - specific siRNA or scrambled ( Scr ) siRNA .

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A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag-USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19-specific siRNA or scrambled (Scr) siRNA.


B Representative images of GFP - LC3B puncta in HeLa - GFP - LC3B cells transfected with USP19 - specific siRNA or scrambled siRNA during growth in normal medium ( DMEM ) or EBSS medium for 3 h . Arrows denote representative autophagosomes . Scale bar , 200 μm .

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B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm.