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 analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. 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 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 analyses; the graph indicate the mean value of the triplicate. All scale bars = 1 µm.

Micronemes and rhoptries are recycled as whole organelles

A) 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). All daughter cells inherited M-MIC2. C) Fluorescence intensity quantification of isolated M-MIC2 signal as illustrated in A. 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). 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. Three biological replicates were used for all analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. 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. C) Quantification of the inheritance of M-SortLR by the daughter cells during replication (stage 1 to 8). All daughter cells inherited M-SortLR. D) Quantification of the signal area of M-SortLR during replication. The total surface of M-SortLR increase at each replication by about a factor 2. Three biological replicates were used for all analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All scale bars = 1 µm.

Degradation of the inner membrane complex.

A) 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. 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. 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 arrow), the cytoplasmic (blue arrow) and the basal (magenta arrow). F) Fluorescence intensity analysis of the three regions defined in E. The curve and analysed region arrow 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 analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. 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; bars represent means ± SD. Scale bars = 1 µm. Myosin-F drives F-actin mediated recycling of maternal organelles via the residual body.

MyoF is essential for recycling apical secretory organelles.

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; bars show means ± SD. All p-values ≤ 0.001 (***), using one-tailed unpaired Student’s t-test. 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) Close-up of heterogeneous M-MIC2 redistribution post-auxin washout. 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). 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. Scale bars = 1 µm.

Schematic 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.

ANKER1 is a resident of the ER.

A) Representative picture of the colocalization ANKER1-Halo with HDEL-GFP transfected parasites. Magenta: ANKER1-Halo, Green: HDEL-GFP. B) Quantification of the colocalization between ANKER1-Halo and HDEL-GFP.

Three biological replicates were used for all analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All scale bars = 1 µm.

Rhoptries are inherited intact.

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 analyses; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All scale bars = 1 µm.

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

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. 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. C) Representative image of a late-stage vacuole shows uniform M-MIC2 distribution among all daughter cells. Scale bar = 5 µm. Three biological replicates were used; bars show means ± SD.

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%. G) IMC intensity in daughters relative to size. Peak intensity occurs when daughters reach ∼3 µm, just before emergence. H) Representative images of daughter cells at different sizes used for classification. Three biological replicates were used; bars show means ± SD. Scale bars = 1 µm.

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

A) 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; bars show means ± SD. Scale bars = 1 µm.

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

A) 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; error bars are standard deviations, and the centre measurement of the graph bars is the mean. All scale bars = 1 µm.