EMBO/SourceData huggingface dataset:
Figure 6Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activityLung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activityMelanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activityGlioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blottingNumber of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructsxenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructsRepresentative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unitShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.
Figure 1Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm.TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.
Figure 2Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm.
Figure 3Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.
Figure 4Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM.
Figure 5Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line.Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites.Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).
Figure 6The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.
Figure 7Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).
Figure 1(C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A).(D) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.
Figure 2(A) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.(B) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, p<0.05 for Ch9 and Ch14).
Figure 3Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (B) Ch14. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (C) Ch9 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.
Figure 4Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch12 (A). All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch10 (B) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch14 (C) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.
Figure 5(A) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.0005 for Ch9, p<0.05 for Ch10 and p<0.005 for Ch14).(B) Tumors sections were stained with H&E to assess tumor histology (scale bars denote 5mm for the inset and 200μm for the zoomed image) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue.(C) Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD p<0.0005 for Ch10 and p<0.005 for Ch14).
Figure 6(A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.005 for Ch10 and p<0.0001 for Ch14). (See also Appendix Figure S3A)(B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9).(C) Quantification of growth in ultra-low adherent, sphere forming conditions for the 4 ICL lines (n=3 for Ch10 and Ch14, n=6 for Ch9 and Ch12 and error bars denote SD, p<0.0001 for Ch9, p<0.001 for Ch10 and p<0.05 for Ch12 and Ch14).(D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.
Figure 7(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.(B) Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes, that have modal chromosome number >2N or ~2N (top, p<0.0001 for chromosome 1-22, X and p<0.05 for chromosome Y; bottom, p<0.0001 for all chromosomes).(C) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome, that have modal chromosome number >2N or ~2N (p<0.0001 for both modal number >2N and ~2N).
Figure 8(A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD).(B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B)(C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).
Figure 1(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments. (E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.
Figure 2U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).
Figure 3(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.