Human WAS patient CD4 + T cells - APC conjugates display synapse symmetry defects similar to those observed in mouse WASP - / - CD4 + T cells . CD4 + T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5 ' ( APC ; see ' Methods ' ) and imaged using confocal microscopy . Each image represents a maximum intensity projection of the synaptic area . Graph in the middle shows values normalized to the mean value of the ' Healthy ' case . In the case of ' actin ' and ' actin foci ' , n = 87 for healthy and n = 63 for WAS ; in the case of pCasL , n = 56 for healthy and n = 21 for WAS ; for ' AR ' graph , n = 88 for healthy and n = 63 for WAS case . P values ; * * * P < 0 . 001 ; n . s . P = 0 . 09 , as determined by using Mann - Whitney two - tailed test between populations of cells within the same experiment .

B-ORGANISM B-DISEASE B-ORGANISM B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-DISEASE B-ORGANISM O O O O O B-CELL_LINE O O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-SUBCELLULAR O 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-GENEPROD O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human WAS patient CD4+T cells- APC conjugates display synapse symmetry defects similar to those observed in mouse WASP-/- CD4+ T cells. CD4+T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5' (APC; see 'Methods') and imaged using confocal microscopy. Each image represents a maximum intensity projection of the synaptic area. Graph in the middle shows values normalized to the mean value of the 'Healthy' case. In the case of 'actin' and 'actin foci', n= 87 for healthy and n= 63 for WAS; in the case of pCasL, n=56 for healthy and n= 21 for WAS; for 'AR' graph, n= 88 for healthy and n= 63 for WAS case. P values; ***P <0.001; n.s. P=0.09, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Simulation of T cell IS F - actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface , using Brownian dynamics equation with no inertia ( see ' Methods ' ) . The images show simulation snapshots at the beginning ( 0 min ; soon after the attachment and spreading of T cells on the substrates ) and the end of the simulations for the three IS cases - with persistent foci ( WT , left most panels ) , with no foci ( WASP - / - , middle right panels ) , and with WASP and consequently foci downregulated after a period of synapse maturation ( WT transitional ) . The colors in the images indicate myosin II motor ( pseudocolored teal ) , F - actin crosslinking proteins ( pseudocolored yellow ) , and the F - actin filaments ( pseudocolored blue for the low tension and the red for thigh tension actin filaments ) ; middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification .

O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O

Simulation of T cell IS F-actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface, using Brownian dynamics equation with no inertia (see 'Methods'). The images show simulation snapshots at the beginning (0 min; soon after the attachment and spreading of T cells on the substrates) and the end of the simulations for the three IS cases - with persistent foci (WT, left most panels), with no foci (WASP-/-, middle right panels), and with WASP and consequently foci downregulated after a period of synapse maturation (WT transitional). The colors in the images indicate myosin II motor (pseudocolored teal), F-actin crosslinking proteins (pseudocolored yellow), and the F-actin filaments (pseudocolored blue for the low tension and the red for thigh tension actin filaments); middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification.


Predicted stress ( tension normalized to the area ) profiles within the cytoskeletal architecture as the synaptic contact progresses in time , corresponding to the three cases shown in ( D ) .

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Predicted stress (tension normalized to the area) profiles within the cytoskeletal architecture as the synaptic contact progresses in time, corresponding to the three cases shown in (D).


Live imaging of a T cell using SIM shows the inter - foci connections ( arrows in the inset ) as predicted in the simulations ; and their loss concomitant with loss of actin foci , as the T cell polarizes to initiate motility ( compare insets between 0 and 120 sec ) . ' 0 sec ' refers to the beginning of the observation of the cell , after it has attached to the substrate , spread and maintained synapse for 12 min . Scale bar , 5µm in main images and 1 . 5μm in the magnified insets .

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Live imaging of a T cell using SIM shows the inter-foci connections (arrows in the inset) as predicted in the simulations; and their loss concomitant with loss of actin foci, as the T cell polarizes to initiate motility (compare insets between 0 and 120 sec). '0 sec' refers to the beginning of the observation of the cell, after it has attached to the substrate, spread and maintained synapse for 12 min. Scale bar, 5µm in main images and 1.5μm in the magnified insets.


Ultrastructural visualization of detergent extracted WT and WASP - / - T cell synaptic cytoskeleton using platinum replica electron microscopy ( top images ) . Each image shows individual synapse , dark zone in the middle of the synapse represents cell nucleus . Scale bar , 2 . 5μm . The bottom left micrograph is the magnified inset from WT synapse and shows actin foci ( identified as globular cluster of short filaments in the inset , marked with arrows ) - dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model , and is lacking in the WASP - / - cell synapse ( bottom right micrograph - magnified inset from WASP - / - synapse ) . Scale bars , 1μm . This experiment was repeated at least twice with similar results .

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Ultrastructural visualization of detergent extracted WT and WASP-/- T cell synaptic cytoskeleton using platinum replica electron microscopy (top images). Each image shows individual synapse, dark zone in the middle of the synapse represents cell nucleus. Scale bar, 2.5μm. The bottom left micrograph is the magnified inset from WT synapse and shows actin foci (identified as globular cluster of short filaments in the inset, marked with arrows)-dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model, and is lacking in the WASP-/- cell synapse (bottom right micrograph- magnified inset from WASP-/- synapse). Scale bars, 1μm.This experiment was repeated at least twice with similar results.


Schematic of the cytoskeletal inhibitors and their targets used in the experiments . SIM imaging of mouse T cells activated for 5 ' on APS , then treated with the indicated inhibitors or vehicle control . Shown are the representative SIM images ( B ) , and quantification of actin foci ( from the phalloidin images ) , talin , or pCasL in the contact interface ( normalized to the mean value in the control case , C ) , and measured cell morphology aspect ratios ( D , E ) . Since CK666 treatment reduces F - actin intensity Kumari et al . , 2015 ( ) , the ' actin ' image of the CK666 treated cell in ( B ) was contrasted differently than the ' control ' and ' Blebb . ' cases for a clearer visualization of F - actin . p values , * P = 0 . 02 , * * P = 0 . 001 , n . s . > 0 . 05 . For the rest of the comparisons between the control and treatment cases , p < 0 . 0001 . For the bar graph in ( B ) , n for foci groups are 214 , 83 , 92 , 98 ; for pCasL groups are 100 , 68 , 91 , 92 ; and for Talin groups are 102 , 70 , 89 , 93 respectively . p value for the n . s . cases is > 0 . 05 , * P = 0 . 02 . In the foci , pCasL and talin groups , when not indicated , the p values are < 0 . 0001 using Mann - Whitney two - tailed test . Central values in the graphs represent Mean and error bars represent ± SEM . These experiments were repeated at least thrice with similar results . Scale bar , 5µm .

O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-SUBCELLULAR O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR 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-GENEPROD O O O O O O O O O O O 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-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic of the cytoskeletal inhibitors and their targets used in the experiments. SIM imaging of mouse T cells activated for 5' on APS, then treated with the indicated inhibitors or vehicle control. Shown are the representative SIM images (B), and quantification of actin foci (from the phalloidin images), talin, or pCasL in the contact interface (normalized to the mean value in the control case, C), and measured cell morphology aspect ratios (D, E). Since CK666 treatment reduces F-actin intensity Kumari et al., 2015(), the 'actin' image of the CK666 treated cell in (B) was contrasted differently than the 'control' and 'Blebb.' cases for a clearer visualization of F-actin. p values, *P =0.02, **P =0.001, n.s. >0.05. For the rest of the comparisons between the control and treatment cases, p <0.0001. For the bar graph in (B), n for foci groups are 214, 83, 92, 98; for pCasL groups are 100, 68, 91, 92; and for Talin groups are 102, 70, 89, 93 respectively. p value for the n.s. cases is > 0.05, *P =0.02. In the foci, pCasL and talin groups, when not indicated, the p values are <0.0001 using Mann-Whitney two-tailed test. Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.


Simulation of synaptic F - actin network following localized myosin inactivation and inactivated myosin - mediated F - actin cross - linking within the area marked with dotted line in the left image . The graph shows the predicted alteration in mechanical stress profile , as myosin is inactivated crosslinked in the defined region ( marked by an arrow ) , ( G ) .

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Simulation of synaptic F-actin network following localized myosin inactivation and inactivated myosin-mediated F-actin cross-linking within the area marked with dotted line in the left image. The graph shows the predicted alteration in mechanical stress profile, as myosin is inactivated crosslinked in the defined region (marked by an arrow), (G).


Experimental confirmation of localized actomyosin crosslinking and symmetry breaking . LifeAct - GFP expressing T cells were incubated with activating substrates , treated with vehicle control or 1µM Azido - blebb and their contact interface imaged using low excitation laser power ( 0 . 5 mW ) , using a spinning disc confocal microscope ( H ) . The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido - Blebb and actomyosin network within the illuminated area ( shown with a box in the image ) . The deflection in center of mass ( COM ) within a minute was calculated and plotted for at least 10 cells in each case ( graphs in I ) . The left graphs in ( I ) show individual COM trajectory , and the graph on the right shows average COM movement of cells within a minute post photo - inactivation of myosinII . This experiment was repeated twice with similar results . Scale bar , 5µm .

O O O B-EXP_ASSAY B-SUBCELLULAR O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O

Experimental confirmation of localized actomyosin crosslinking and symmetry breaking. LifeAct-GFP expressing T cells were incubated with activating substrates, treated with vehicle control or 1µM Azido-blebb and their contact interface imaged using low excitation laser power (0.5 mW), using a spinning disc confocal microscope (H). The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido-Blebb and actomyosin network within the illuminated area (shown with a box in the image). The deflection in center of mass (COM) within a minute was calculated and plotted for at least 10 cells in each case (graphs in I). The left graphs in (I) show individual COM trajectory, and the graph on the right shows average COM movement of cells within a minute post photo-inactivation of myosinII. This experiment was repeated twice with similar results. Scale bar, 5µm.


T cells were activated using Alexa568 - 2C11 and ICAM1 reconstituted lipid bilayers for 5 min , fixed , stained with phalloidin and imaged using SIM . In the graph , n = 50 for WT and 61 for WASP - / - , P value , P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test . The graphs in ( B ) show synaptic levels of indicated proteins , normalized to the mean levels of ' + WASP ' in each case . For the left graph , n = 48 , 46 , 52 , 46 respectively , and p value , P * = 0 . 03 . For the right graph , n = 50 for WT and n = 61 for WASP - / - in ' Actin ' , ' Foci ' , as well as ' pCasL ' cases . P values P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test .

B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

T cells were activated using Alexa568-2C11 and ICAM1 reconstituted lipid bilayers for 5 min, fixed, stained with phalloidin and imaged using SIM. In the graph, n= 50 for WT and 61 for WASP-/-, P value, P***<0.0001, obtained using Mann-Whitney two-tailed test. The graphs in (B) show synaptic levels of indicated proteins, normalized to the mean levels of '+WASP' in each case. For the left graph, n= 48, 46, 52, 46 respectively, and p value, P *= 0.03. For the right graph, n= 50 for WT and n= 61 for WASP-/- in 'Actin', 'Foci', as well as 'pCasL' cases. P values P*** <0.0001, obtained using Mann-Whitney two-tailed test.


Synapse breaking behavior of T cells on hydrogels of indicated stiffness , covalently conjugated with 2C11 and ICAM1 . These experiments were repeated at least thrice with similar results . In the left graph , n for WT = 14 , for WASP - / - , n = 19 ; P * * = 0 . 0015 ; in the graph on the right , n for WT = 40 , n for WASP - / - = 36 , P * * * = 0 . 0006 , as assessed using Mann - Whitney two - tailed test .

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Synapse breaking behavior of T cells on hydrogels of indicated stiffness, covalently conjugated with 2C11 and ICAM1. These experiments were repeated at least thrice with similar results. In the left graph, n for WT= 14, for WASP-/-, n= 19; P**= 0.0015; in the graph on the right, n for WT= 40, n for WASP-/-= 36, P***= 0.0006, as assessed using Mann-Whitney two-tailed test.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) together with GFP - RAB11 to visualize trafficking of recycling endosomes ( RE ) . At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport . Representative dendrite segments and kymographs of GFP - RAB11 vesicle movement . Quantitative analysis of vesicle motility ( B ) and total number ( C ) from kymographs .

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(A) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs.


( D ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . TDP - 43 levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 .

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(D) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1.


( E ) iPSC - derived human neurons ( DIV7 + 3 ) were transduced with shTDP or shCtrl and GFP - RAB11 and at least three dendrite segments per neuron analyzed as in ( a ) .

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(E) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a).


Quantitative analysis of vesicle motility ( F ) and number ( G ) from kymographs . Mean + / - s . e . m . , n = 3 , unpaired two - tailed t - test : * denotes p < 0 . 05 , * * denotes p < 0 . 01 , * * * denotes p < 0 . 001 .

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Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 or a control together with tagRFP ( not depicted ) . Representative images of neurons subjected to Alexa - transferrin ( Alexa - Tf ) for 20 min ( pulse ) and chased for 0 , 20 or 60 min in complete media . Scale bar represents 100 µm . ( B ) Quantification of labeled cellular Alexa - Tf normalized to cell area ( using tagRFP ) after chase period . At least ten images per condition were analyzed per experiment in three independent experiments .

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(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 or a control together with tagRFP (not depicted). Representative images of neurons subjected to Alexa-transferrin (Alexa-Tf) for 20 min (pulse) and chased for 0, 20 or 60 min in complete media. Scale bar represents 100 µm. (B) Quantification of labeled cellular Alexa-Tf normalized to cell area (using tagRFP) after chase period. At least ten images per condition were analyzed per experiment in three independent experiments.


( C ) ELISA measurement of β2 - transferrin in CSF of control subjects ( n = 65 ) and sporadic ALS patients ( n = 55 ) , FTD - ALS patients ( n = 12 ) and behavioral variant FTD patients ( bvFTD , n = 16 ) .

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(C) ELISA measurement of β2-transferrin in CSF of control subjects (n=65) and sporadic ALS patients (n=55), FTD-ALS patients (n=12) and behavioral variant FTD patients (bvFTD, n=16).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected shTDP or shCtrl and GFP as transfection marker . After 3 days neurons were subjected to Alexa - Tf for 20 min and intracellular Alexa - Tf movement was imaged as in Figure 1 afterwards . Quantitative analysis of Alexa - Tf movement ( E ) and vesicle number ( F ) from kymographs .

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(D) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transfected with myc - tagged TDP - 43 wild - type or a mutant lacking the nuclear localization signal ( ΔNLS ) or an empty vector control . Immunostaining with the indicated antibodies and DAPI to label nuclei . Scale bar represents 50 µm .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O

(A) Primary hippocampal neurons (DIV6+4) were transfected with myc-tagged TDP-43 wild-type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 µm.


( B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either TDP - 43 wild - type , TDP - 43ΔNLS or an empty vector control together with GFP - RAB11 to visualize recycling endosomes and analyzed as in Figure 1 . Quantitative analysis of recycling endosome movement ( C ) and vesicle number ( D ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

(B) Primary hippocampal neurons (DIV6+3) were transfected with either TDP-43 wild-type, TDP-43ΔNLS or an empty vector control together with GFP-RAB11 to visualize recycling endosomes and analyzed as in Figure 1. Quantitative analysis of recycling endosome movement (C) and vesicle number (D).


( A ) Primary cortical neurons were transduced with shTDP or control ( DIV2 + 7 ) and subjected to quantitative proteomic analysis . Volcano plot shows up and down regulated proteins from three biological replicates . Significant changes are colored . Compare Table EV1 .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O 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) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1.


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . Immunoblots with the indicated antibodies ( C ) . Quantification of VPS4B and TDP - 43 protein level using densitometry ( n = 4 ) . Expression was normalized to the housekeeping gene β - actin ( D ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D).


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . VPS4B mRNA level were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping genes YWHAZ and PGK1 ( E ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O

(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E).


( F ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either shTDP or shCtrl and iRFP670 . Immunostaining with the indicated antibodies . Scale bar represents 50 µm .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm.


( G ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . VPS4B levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 ( n = 3 ) .

O O O B-CELL_TYPE O O B-ORGANISM B-CELL_TYPE O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).


( A - B ) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP - 43 . ( B ) HEK293 cells were transfected with shRNAs targeting human TDP - 43 , a control shRNA , TDP - 43 wild - type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter . VPS4B promoter - driven renilla luciferase activity was normalized to TK promoter - driven firefly luciferase . Quantification from six independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O

(A-B) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP-43. (B) HEK293 cells were transfected with shRNAs targeting human TDP-43, a control shRNA, TDP-43 wild-type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter-driven renilla luciferase activity was normalized to TK promoter-driven firefly luciferase. Quantification from six independent experiments.


( C - D ) ChIP assay to analyze binding of TDP - 43 to VPS4B promoter region in rat cortical neurons , HEK293 cells and human brain tissue . PCR reaction from input , negative control and TDP - 43 immunoprecipitates . Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O O B-ORGANISM B-TISSUE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) ChIP assay to analyze binding of TDP-43 to VPS4B promoter region in rat cortical neurons, HEK293 cells and human brain tissue. PCR reaction from input, negative control and TDP-43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input.


( E - H ) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD / ALS - TDP and five healthy controls . ( E , G ) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord . Scale bar represents 50 µm . ( F , H ) Quantification of cellular VPS4B levels . At least 50 cells per case were analyzed .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O 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-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(E-H) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD/ALS-TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 µm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either VPS4B or an empty vector control together with GFP - RAB11 to visualize recycling endosomes . Neurons were imaged as in Figure 1 to analyze recycling endosome transport . Quantitative analysis of vesicle motility ( B ) and number ( C ) from kymographs ( n = 4 ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with the indicated combinations of shRNA targeting TDP - 43 , VPS4B and control together with GFP - RAB11 and imaged as in Figure 1A . Quantitative analysis of vesicle motility ( E ) and number ( F ) from kymographs ( n = 3 ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

(D) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3).


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification . Volcano plot depicts proteins that are significantly ( p < 0 . 01 , t - test , Benjamini - Hochberg , FDR : 0 . 05 ) decreased or increased on the cell surface in three independent experiments with two technical replicates . For full dataset see Table EV2 .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2.


( B - C ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the ErbB4 ligand NRG1 ( 10 nM ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( B ) . Quantification of total and phosho - ERBB4 ( Y1056 ) using densitometry 3 - 7 biological replicates ( C ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(B-C) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B). Quantification of total and phosho-ERBB4 (Y1056) using densitometry 3-7 biological replicates (C).


( D - E ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the FGFR1 ligand FGF1 ( 10 ng / ml ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( D ) . Quantification of total and phosho - FGFR1 ( Y654 ) using densitometry 3 - 7 biological replicates ( E ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(D-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (D). Quantification of total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (E).


( F ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shTDP or shCtrl . Gene expression was analyzed by quantitative RT - PCR normalized to the housekeeping genes YWHAZ and GAPDH ( n = 3 )

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O

(F) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3)


( G ) Hippocampal neurons were transfected with ErbB4 containing a luminal HA - tag ( HA / T - ErbB4 ) and shCtrl or shTDP ( DIV9 + 3 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with HA - antibodies . ( H ) Quantification of HA / T - ErbB4 surface levels . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD 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) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


( A , B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shCtrl or shTDP and ErbB4 containing an N - terminal HA epitope tag and followed by thrombin cleavage site ( HA / T - ErbB4 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with two different HA - antibodies . After removing the HA - signal of surface ErbB4 ( 0 min ) using thrombin cleavage ( 5min , 1U / ml ) membrane insertion of HA / T - ErbB4 was measured at the indicated time points . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O 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,B) Primary hippocampal neurons (DIV6+3) were transfected with shCtrl or shTDP and ErbB4 containing an N-terminal HA epitope tag and followed by thrombin cleavage site (HA/T-ErbB4). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with two different HA-antibodies. After removing the HA-signal of surface ErbB4 (0 min) using thrombin cleavage (5min, 1U/ml) membrane insertion of HA/T-ErbB4 was measured at the indicated time points. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP 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 . ( A , B ) TDP - 43 knockdown ( DIV6 + 5 ) significantly reduces dendrite branching compared to control : at 25 , 37 . 5 and 87 . 5 µm radius p < 0 . 05 , at 50 µm p < 0 . 01 and from 62 . 5 to 75 µm p < 0 . 001 .

O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 shTDP 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. (A, B) TDP-43 knockdown (DIV6+5) significantly reduces dendrite branching compared to control: at 25, 37.5 and 87.5 µm radius p < 0.05, at 50 µm p < 0.01 and from 62.5 to 75 µm p < 0.001.


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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O 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-GENEPROD 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 .

B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O O B-TISSUE O O O O O 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-GENEPROD O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-TISSUE O O O O O 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-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O 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-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY 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 .

B-EXP_ASSAY O O O O B-EXP_ASSAY B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-TISSUE I-TISSUE O 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 ) .

B-EXP_ASSAY O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 .

B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE I-TISSUE 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 .

B-EXP_ASSAY O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR 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 B-EXP_ASSAY O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O 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-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O 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-CELL_TYPE O B-CELL_TYPE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD B-EXP_ASSAY 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 B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-TISSUE O O O O O B-CELL_TYPE O O O O O O O O O O O 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-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE 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 .

O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-TISSUE O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY O

(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 .

B-EXP_ASSAY O O O O B-GENEPROD O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O

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 .

O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-TISSUE O O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O

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 .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O

(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 .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

(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 .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O B-TISSUE I-TISSUE O O B-ORGANISM O O B-SUBCELLULAR O O B-ORGANISM O O B-SUBCELLULAR O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

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 .

O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-ORGANISM O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

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 .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-ORGANISM O O B-EXP_ASSAY O B-ORGANISM O B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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. 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 .

O O O O B-ORGANISM I-ORGANISM B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-EXP_ASSAY O O

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 .

O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O B-ORGANISM O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-ORGANISM O O O O O O O O O O O O O O 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. 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 .

O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O B-ORGANISM O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 :

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O O O

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

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O 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. 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 :

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-GENEPROD B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O B-ORGANISM B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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 .

O O O B-EXP_ASSAY O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O 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. 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.