Discrimination between maternal and de novo material.

A) Dual staining scheme. Maternal material is labelled initially with Janila Fluor®-646, and the excess washed out. After replication of parasites de novo synthesised material is labelled with Janila Fluor®-549, allowing efficient discrimination of the two populations. B) Fluorescence intensity quantification of maternal (M) and de novo sythenetised (n) MIC2 and IMC1 during replication from stage 1 to 8. Magenta: M-MIC2, Rosa: M-IMC1, Green: n-MIC2, Dark Green: n-IMC1. C) Representative picture of MIC2-Halo parasites during replication (stage 1 to 8) stained for both maternal (M) and de-novo (n) MIC2. Magenta: M-MIC2, Green: n-MIC2. Maternal MIC2 is efficiently recycled into the daughters, while de novo MIC2 is formed after each replication cycle D) Representative picture of IMC1-Halo parasites during replication (stage 1 to 8) stained for both maternal (M) and de-novo (n) IMC1. Magenta: M-IMC1, Green: n-IMC1. In contrast to micronemes, maternal IMC is degraded, and only residual amount is detected after the first replication cycle. Three biological replicates were used for all analysis with a total of 300 IMC-1 Halo vacuoles and 260 MIC2-Halo vacuoles analysed; error bars are standard deviations, and the centre measurement of the graph bars is the mean. Panels C and D show maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Three recycling fates for the maternal organelles.

A) Overview of T. gondii organelles and molecular markers used for visualisation with endogenous Halo-Tags. B) Fluorescence intensity (FI) quantification of maternal (M) molecular marker listed in A. The fluorescence intensity variation during replication, allows to group them into three groups: 1) Efficient, almost quantitative recycling. 2) even distribution of maternal material and 3) almost exclusive de novo synthesis. Dark blue: RON2, Blue: ROP1, Pale blue: MIC2, Dark green: SortLR, Green: MyoA, Pale green: Tic20, Greenish white: ANKER1, Gold: GAPM1a, Yellow: IMC1. C) Representative picture of parasites expressing indicated Halo tagged proteins (stage 1 to 8) stained for maternal (M) proteins. Three biological replicates were used for all analysis with an average of 265 vacuoles analysed per protein; the graph indicate the mean value of FI calculated from the triplicate. Panels C shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Micronemes and rhoptries are recycled as whole organelles

Representative picture of MIC2-Halo during replication (stage 1 to 8) stained for both the maternal (M) and de novo (n) MIC2. Magenta: M-MIC2, Green: n-MIC2. Zoom windows allow to visualise isolated apical M-MIC2 signal for which the fluorescence intensity is quantified in C. B) Quantification of the inheritance of M-MIC2 by the daughter cells during replication (stage 1 to 8). A total of 260 vacuoles were analysed. All daughter cells inherited M-MIC2. C) Fluorescence intensity quantification of isolated M-MIC2 signal as illustrated in A. A total of 203 isolated M-MIC2 signals were analysed. D) Representative picture of RON2-Halo during replication (stage 1 to 8) stained for both the maternal (M) and de novo (n) MIC2. Magenta: M-RON2, Green: n-RON2. Zoom windows allow to visualise isolated apical M-RON2 signal for which the fluorescence intensity is quantified in F. E) Quantification of the inheritance of M-RON2 by the daughter cells during replication (stage 1 to 8). A total of 289 vacuoles were analysed. Not all daughter cells inherit M-RON2 (asterisks), best seen in later replication stages. F) Fluorescence intensity quantification of isolated M-RON2 signal as illustrated in D. A total of 215 isolated M-RON2 signals were analysed. Three biological replicates were used for all analysis; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A and D show maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Expansion of the mother organelle for equivalent sharing to the daughter.

A) Representative picture of SortLR-Halo during replication (stage 1 to 8) stained for both the maternal (M) and de novo (n) SortLR. Magenta: M-SortLR, Green: n-SortLR. B) Fluorescence intensity quantification of M-SortLR and n-SortLR illustrated in A. Magenta: M-SortLR, Green: n-SortLR. A total of 251 vacuoles were analysed. C) Quantification of the inheritance of M-SortLR by the daughter cells during replication (stage 1 to 8). All daughter cells inherited M-SortLR. A total of 100 vacuoles were analysed. D) Quantification of the signal area of M-SortLR during replication. A total of 100 vacuoles were analysed. The total surface of M-SortLR increase at each replication by about a factor 2. Three biological replicates were used for all analysis; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Degradation of the inner membrane complex.

Representative picture of GAPM1a-Halo during replication (stage 1 to 8) stained for both the maternal (M) and de novo (n) GAPM1a. B) Fluorescence intensity quantification of M-GAPM1a and n-GAPM1a illustrated in A. A total of 265 vacuoles were analysed. C) Quantification of the inheritance of M-GAPM1a by the daughter cells during replication (stage 1 to 8). After replication, M-GAPM1a is not visible in daughter cells. A total of 265 vacuoles were analysed. *The calculation was performed using single stage parasite intensity to set up the exposure as performed for all the other organelles. Under those conditions the remaining signal of the maternal GAPM1a is below the detection level and cannot be observed. D) GAPM1a is degraded in the RB. Representative images of GAPM1a-Halo parasites at different stages of daughter cell development. M-GAPM1a collapses toward the forming residual body, where the signal disappears after completion of replication, indicating it’s degradation. E) Time series of IMC1-Halo parasites during the first replication. Parasite were stained for the maternal (M) IMC1 prior invasion. Three regions of the parasites were analysed: the apical (green ROI), the cytoplasmic (blue ROI) and the basal (magenta ROI). F) Fluorescence intensity analysis of the three regions defined in E. The curve and analysed ROI share the same colour code. Green: Apical, Blue: Cytoplasm, Magenta: Basal. After the accumulation of the mother IMC at the basal pole, the FI of M-IMC1 decrease without redistributing to any other region. Three biological replicates were used for all analysis; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A and D show maximum-intensity projections of Z-stack images. Panel E displays single optical plane images acquired during live imaging. All scale bars = 1 µm.

Micronemes and rhoptries are recycled similarly, but differ in timing and location.

A) Time-lapse of MIC2-Halo over two replication cycles. Maternal MIC2 (M-MIC2) is stained, and daughter cell formation is visualized via IMC1-YFP. M-MIC2 is transported via the residual body (RB) (white arrows; see Video S2). B) Time-lapse of RON2-Halo parasites with IMC-YFP. Maternal RON2 (M-RON2) is transported before mother cell collapse and RB formation (white arrows). C) Representative images showing maternal organelles associating with F-actin and the RB. Insets highlight colocalization of maternal proteins and F-actin. D) Quantification of M-MIC2 and M-RON2 colocalization with F-actin. Data from three biological replicates, a total of 534 RON2-Halo and 342 MIC2-Halo vacuoles were analysed; bars represent means ± SD. Panel A and B display single optical plane images acquired during live imaging. Panel C show single Z plane of Z-stack images. Scale bars = 1 µm.

Myosin-F drives F-actin mediated recycling of maternal organelles via the residual body.

A) Effect of MyoF knockdown (KD) on maternal microneme inheritance. MyoF-mAID MIC2-Halo parasites were labeled and grown ± auxin for 24h. Magenta: maternal MIC2 (M-MIC2), Green: newly synthesized MIC2 (n-MIC2). Without MyoF, M-MIC2 accumulates in the residual body (RB) instead of being passed to daughter cells (Stage 1–2), with increasing accumulation over replication cycles (Stage 2–8). B) Effect of MyoF KD on maternal rhoptry inheritance. MyoF-mAID RON2-Halo parasites show M-RON2 retention in the RB, mirroring the pattern seen with MIC2. C) Effect of MyoF KD on maternal Golgi inheritance. MyoF-mAID SortLR-Halo parasites show no significant RB accumulation of M-SortLR, unlike MIC2 and RON2. D) Quantification of vacuoles showing RB accumulation. White: control; Gray: MyoF-KD. Three biological replicates were used; a total of 675 Ctrl and 743 KD vacuoles were analyzed for MIC2-Halo, 645 Ctrl and 594 KD for RON2-Halo and 795 Ctrl and 619 KD for SortLR-Halo, bars show means ± SD. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A, B and C show maximum-intensity projections of Z-stack images. Scale bars = 1 µm

The residual body (RB) functions as a recycling center, not a dead end.

To assess the fate of material accumulated in the RB, auxin chase experiments were performed: 24h with auxin followed by 24h without. A) Representative images of MyoF-mAID MIC2-Halo parasites after 48h in control (no auxin), continuous auxin (Aux), or auxin washout (Aux washed). Magenta: M-MIC2, Green: n-MIC2. Continuous auxin led to M-MIC2 accumulation in the RB, while auxin washout enabled redistribution. B) Representative images of MyoF-mAID MIC2-Halo parasites after 48h in control (no auxin), continuous auxin (Aux), or auxin washout (Aux washed). Magenta: M-MIC2, Green: α-HA (MyoF). In absence of auxin MyoF is visible, induction with auxin deplete MyoF led to M-MIC2 accumulation in the RB, while auxin washout enabled MyoF production and M-MIC2 redistribution. C) Quantification of vacuoles showing normal, accumulated, or redistributed M-MIC2. White: control; Gray: MyoF-KD; Blue: MyoF-KD auxin chase. D) Time-lapse without auxin: M-MIC2 briefly passes through the RB (white arrows). A total of 170 Ctrl, 186 KD and 179 auxin chased vacuoles were analyzed. E) Time-lapse with auxin: sustained M-MIC2 accumulation in the RB throughout replication. F) Time-lapse after auxin washout: initial M-MIC2 accumulation in the RB resolves by the second replication cycle, with redistribution observed. Three biological replicates were used; bars show means ± SD. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A and B show maximum-intensity projections of Z-stack images. Panel D, E and F display single optical plane images acquired during live imaging. Scale bars = 1 µm.

Schematic summaries.

A: Summary of the three fates of the maternal organelles. Group 1: Efficient, intact organelle recycling. De novo organelles are generated independently of the maternal organelle but in a similar location. Both de novo and maternal are distributed to the daughter cells. The Fluorescence intensity of the maternal organelle is relatively stable. Group 2: Even distribution of maternal material through organelle expansion with the insertion of the de novo material in the mother organelle. The Fluorescence intensity of the maternal organelle is divided by two at each replication step but their signal surface is increased by two. Group 3: Almost exclusive de novo synthesis. The de novo organelle is generated independently of the maternal organelle and in a different location. The maternal organelle is not clearly observable after a cycle of replication. The fluorescence intensity of the maternal organelle drastically drops after the first replication. Magenta: maternal organelle, Green: de novo organelle, Yellow: Colocalization between maternal and de novo. B: Summary of the MyoF regulated inheritance. The maternal and the de novo micronemes are generated independently. In presence of MyoF (F1), the daughter cells form a chimera possessing both maternal and de novo micronemes, which are distributed in a relatively equal manner even after multiple replication cycles. In absence of MyoF, (F2) the inherited micronemes accumulate in the residual body and the daugthers cells are mostly composed of de novo micronemes. If the depletion of MyoF is maintained (F2.1) more and more inherited micronemes accumulate inside the residual body without important degradation. In opposition, if the expression of the MyoF is reestablished by the auxin removal (F3), the inherited micronemes stuck inside the residual body are redistributed but in a less equal manner than usually observed for control.

Fluorescence intensity of maternal and de novo MIC2 and IMC1.

A) Fluorescence intensity quantification of M-MIC2 and n-MIC2 illustrated in Figure 1A, B and presented here independently and with statistical analysis. Magenta: M-MIC2, Green: n-MIC2. B) Fluorescence intensity quantification of M-IMC1 and n-IMC1 illustrated in Figure 1A, B and presented here independently and with statistical analysis. Magenta: M-IMC1, Green: n-IMC1. Three biological replicates were used for all analysis; a total of 300 and 260 vacuoles were analysed for IMC1 and MIC2 respectively, bars show means ± SD. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test.

ANKER1 is a resident of the ER and independent fluorescence analysis from 1B.

A) Representative picture of the colocalization ANKER1-Halo with HDEL-GFP transfected parasites. Magenta: ANKER1-Halo, Green: HDEL-GFP. B) Quantification of the percentage of vacuoles with colocalization between ANKER1-Halo and HDEL-GFP. A total of 169 vacuoles were analysed. Pearson correlation coefficient was also calculated between ANKER-1 and HDEL-GFP from a total 30 independent vacuoles, 0,92±0,02. C) Fluorescence intensity quantification of M-RON2 illustrated in Figure 2B and presented here independently and with statistical analysis. A total of 272 vacuoles were analysed D) Fluorescence intensity quantification of M-ROP1 illustrated in Figure 2B and presented here independently and with statistical analysis. A total of 286 vacuoles were analysed. E) Fluorescence intensity quantification of M-MIC2 illustrated in Figure 1B, 2B and S1A and presented here independently and with statistical analysis. A total of 260 vacuoles were analysed. F) Fluorescence intensity quantification of M-TIC20 illustrated in Figure 1B and presented here independently and with statistical analysis. A total of 223 vacuoles were analysed. G) Fluorescence intensity quantification of M-ANKER1 illustrated in Figure 1 B and presented here independently and with statistical analysis. A total of 284 vacuoles were analysed. H) Fluorescence intensity quantification of M-MyoA illustrated in Figure 1B and presented here independently and with statistical analysis. A total of 276 vacuoles were analysed. I) Fluorescence intensity quantification of M-IMC1 illustrated in Figure 1B, 2B and S1B and presented here independently and with statistical analysis. A total of 300 vacuoles were analysed. J) Fluorescence intensity quantification of M-GAPM1a illustrated in Figure 2B and 5B and presented here independently and with statistical analysis. A total of 265 vacuoles were analysed. All datasets were regrouped here to allow a better visual comparison between conditions. Three biological replicates were used for all analysis; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Rhoptries are inherited intact.

A) The de-novo secretary organelle are generated independently of the maternal organelles. Representative picture of MIC2 and RON2-Halo parasites. Magenta: Maternal, Green: de novo. Zoom window highlight the absence of colocalization between maternal and de novo material. Three biological replicates were used. Panels A shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

The de novo secretory organelles are generated independently of the maternal.

A) Quantification of the average signal area of M-RON2 during replication. The average surface of M-RON2 decrease at each replication suggesting a separation of the mother organelle. B) Quantification of the total signal area of M-RON2 during replication. Despite the decrease of the average signal area, the total surface of M-RON2 remain stable indicating that the totality of the maternal organelles are conserved during replication. C) Fluorescence intensity analysis of n-RON2. D) Representative picture of a vacuole with missing maternal rhoptries in some daughter cell of a single vacuole at stage 8. Magenta: M-RON2, Green: n-RON2. Three biological replicates were used for all analysis; error bars are standard deviations, and the centre measurement of the graph bars is the mean. For each panel A,B,C a total of 289 vacuoles were analysed. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels D shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Maternal micronemes are evenly distributed to daughter cells.

A) MIC2-Halo parasites at stage 8 were stained for maternal (M-MIC2, magenta) and de novo (n-MIC2, green) MIC2. n-MIC2 signal was used to outline each tachyzoite, numbered 1–8 left to right, and M-MIC2 micronemes were counted per cell. Scale bar = 1 µm. B) Quantification of average M-MIC2 micronemes per tachyzoite shows consistent numbers across daughters, indicating equal distribution. A total of 18 vacuoles were analysed for a total of 1341 isolated M-MIC2 signals. C) Representative image of a late-stage vacuole shows uniform M-MIC2 distribution among all daughter cells. D) Supplementary images from the different replicate that illustrating the inheritance pattern is constant between vacuoles. All p-values ≥ 0.05 (ns), using two-tailed unpaired Student’s t-test. Panels A, C and D show maximum-intensity projections of Z-stack images. Scale bar = 5 µm. Three biological replicates were used; bars show means ± SD. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test.

Maternal and de novo signal of the other protein of the group 2.

All parasite lines were labelled for maternal (pior invasion, Jan 646, magenta) and de novo material (after replication, Jan 549, green) A) ANKER1-Halo. Magenta: M-ANKER1, Green: n-ANKER1. B) MyoA-Halo. Magenta: M-MyoA, Green: n-MyoA. C) TIC20-Halo. Magenta: M-TIC20, Green: n-TIC20. Panels displays maximum-intensity projections of Z-stack images. Scale bars = 1 µm.

IMC degradation occurs after daughter cell formation but before budding.

A) Time-lapse of IMC1-Halo during the first replication cycle, with F-actin visualized via chromobody-emerald. Magenta: M-IMC1, Green: F-actin. M-IMC1 accumulates in the residual body with no redistribution or signal loss in non-replicating parasites. B) Fluorescence intensity tracking of parasites 1–4 (see A) during replication of parasite 3. C) Mean fluorescence intensity across apical, cytoplasmic, and basal regions in 25 replicating parasites. D) Mean M-IMC1 intensity in 25 non-replicating parasites during the same timeframe as C. E) Image showing daughter cells forming within the mother. Green: IMC1-YFP. Blue arrow: mother; yellow arrows: daughters. F) IMC fluorescence comparison between mother and daughters, with mother set to 100%. A total of 47 daughter cells and 75 maternal IMC were analysed. G) IMC intensity in daughters relative to size. Peak intensity occurs when daughters reach ∼3 µm, just before emergence. A total of 100 vacuoles were analysed. H) Representative images of daughter cells at different sizes used for classification. Three biological replicates were used; bars show means ± SD. Panel A displays single optical plane images acquired during live imaging. Panels E and H show maximum-intensity projections of Z-stack images. Scale bars = 1 µm.

AMA1 and MIC4 recycling is impaired in the absence of MyoF.

Effect of MyoF knockdown (KD) on AMA1 inheritance. MyoF-mAID MIC2-Halo parasites were grown ± auxin for 24h. Magenta: M-MIC2, Green: n-MIC2, Cyan: α-AMA1. Without MyoF, AMA1 accumulates in the residual body, mirroring the MIC2-Halo pattern. B) Effect of MyoF KD on MIC4 inheritance. Magenta: M-MIC2, Green: n-MIC2, Cyan: α-MIC4. MIC4 also accumulates in the residual body over time in the absence of MyoF. C-D) Quantification of vacuoles showing α-AMA1 (C) and α-MIC4 (D) accumulation. White: control; Gray: MyoF-KD. Three biological replicates were used; a total of 904 Ctrl and 731 KD vacuoles for AMA1 and 691 Ctrl and 661 KD for MIC4 were analysed. bars show means ± SD. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A and B show maximum-intensity projections of Z-stack images. Scale bars = 1 µm.

In absence of MyoF, MIC8 is also blocked in its recycling.

Impact of MyoF KD on MIC8 inheritance during replication. MyoF-mAID MIC2-Halo parasites were labelled and grown for 24h +/- auxin. MIC8 was visualised using antibodies. Magenta: M-MIC2, Green: n-MIC2, Cyan: α-MIC8. In absence of MyoF, as observed for all the other microneme markers, α-MIC8 accumulate as replication goes on, following a similar patten as MIC2-Halo. C) Quantification of the percentage of vacuoles exhibiting accumulation of α-MIC8 in the residual body. White: Control, Gray: MyoF-KD. Three biological replicates were used for all analyses; a total of 900 Ctrl and 671 KD vacuoles were analysed, error bars are standard deviations, and the centre measurement of the graph bars is the mean. All p-values ≤ 0.001 (***), using two-tailed unpaired Student’s t-test. Panels A shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Redistribution of micronemes following auxine washout.

A) Representative images of MyoF-mAID MIC2-Halo parasites after 48h in control (no auxin), continuous auxin (Aux), or auxin washout (Aux washed) showing extreme alteration of the redistribution of the M-MIC2. Magenta: M-MIC2, Green: n-MIC2. B) Quantification of vacuoles showing normal, accumulated, or redistributed and MyoF expression. Blue: normal distribution; Gray: accumulated micronemes; White: uneven distribution, Yellow: MyoF. A total of 99 Crl, 120 Kd and 144 auxin washout vacuoles were analysed. C) Representative picture of a control and a vacuole after auxin washout and microneme redistribution, side panel show a focus on an apical part of one tachyzoite of the vacuole and a focus on an isolated microneme signal. D) Quantification of the M-MIC2 fluorescence intensity between control and auxine washout vacuoles. A total of 150 independent micronemes signal were analyzed for both Ctrl and IAA chased. No significant difference was observed suggesting the absence of major degradation process after the 24h sequestration of the micronemes in the residual body. Three biological replicates were used for all analysis. All p-values ≤ 0.001 (***), all p-values ≥ 0.05 (ns) using two-tailed unpaired Student’s t-test. Panels A and C shows maximum-intensity projections of Z-stack images. All scale bars = 1 µm.

Image processing and fluorescence intensity measurements.

Supplementary information supporting the Materials and Methods describing image processing and region selection used for fluorescence intensity quantification throughout the study. (A) Schematic of the imaging strategy, showing Z-stack acquisition centred on the middle of the vacuole, followed by maximum-intensity projection without intermediate image processing. Maximum-intensity projection was chosen over mean projection as it provides a more conservative measure of fluorescence intensity. (B) Illustration of gray-value measurements obtained from the same region of interest (ROI), comparing maximum and mean gray values. While the maximum gray value is independent of ROI size, the mean gray value varies with ROI area. (C) Examples of ROI selection and exclusion for gray-value measurements in parasites at different replication stages.(D) Example of ROI selection for gray-value measurement of mother versus daughter IMC, using the initial maternal IMC signal to discriminate between the two.