EMBO/SourceData huggingface dataset:
Figure 9Primary 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.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.
Figure 1Confocal 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.
Figure 2Confocal 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.
Figure 4Confocal 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.
Figure 5(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.
Figure 7Confocal 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.
Figure 8(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.
Figure 2A. 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.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:secondsE. 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 measurementsF. 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
Figure 3A. 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.E. Tight Junction assay on fixed samples depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation. Data information: Error bars represent standard deviation. One-way ANOVA analysis was performed for each graph ****p value <0.0001.F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines. One-way ANOVA analysis was performed for each graph ****p value <0.0001.
Figure 4A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads). C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow). Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm.D. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present. F. The posterior end of the parasite also deforms during the invasion process.G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events. Data information: Scale bar represents 5 µm. White arrow points to direction of invasion.
Figure 5A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion.D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.
Figure 6A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparumB. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum
Figure 1(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.
Figure 2(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.MYB protein (C) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting respectively. β-actin served as an internal control. N- normoxia and H- hypoxia.(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.
Figure 3(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.
Figure 4(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.