# Two anchoring proteins control daughter apical complex assembly in Toxoplasma gondii

Mélanie Burette, Marcin Pezinsky, Quynh Nguyen, Alae Benharoual, Sharon Patray, Marjorie Maynadier, Jason Delabre, Thomas Mouveaux, Arnault Graindorge, Laurence Berry, et al.

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Mélanie Burette, Marcin Pezinsky, Quynh Nguyen, Alae Benharoual, Sharon Patray, et al. Two anchoring proteins control daughter apical complex assembly in Toxoplasma gondii. 2026. hal-05516499

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1 **Two anchoring proteins control daughter apical complex assembly in *Toxoplasma gondii***

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3

4 Mélanie Burette¹, Marcin Pezinsky², Quynh Nguyen³,⁴, Alae Benharoual¹, Sharon Patray³,⁴,

5 Marjorie Maynadier¹, Jason Delabre¹, Thomas Mouveaux², Arnault Graindorge¹, Laurence

6 Berry¹, Jean-Francois Dubremetz¹, Valérie Rofidal⁵, Yi-Wei Chang³,⁴, Mathieu Gissot²&

7 Maryse Lebrun¹

8

9 ¹Laboratory of Pathogens and Host Immunity, LPHI, UMR 5294 CNRS, UA15 INSERM,
10 Université de Montpellier, Montpellier 34095, France

11 ²Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-
12 Center for Infection and Immunity of Lille, F-59000 Lille, France

13 ³Department of Biochemistry and Biophysics, Perelman School of Medicine, University of
14 Pennsylvania, Philadelphia 19104, USA

15 ⁴Institute of Structural Biology, Perelman School of Medicine, University of Pennsylvania,
16 Philadelphia 19104, USA

17 ⁵IPSiM, Univ. Montpellier, CNRS, INRAE, Institut Agro, France

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19 corresponding authors: maryse.lebrun@umontpellier.fr ; mathieu.gissot@pasteur-lille.fr

20

21 **Abstract**

22 During *Toxoplasma gondii* division, the apical complex—comprising the conoid, apical polar

23 ring (APR), and preconoidal rings—assembles with precise spatiotemporal coordination to

24 form functional daughter buds. Despite their essential roles in invasion, motility, and division,

25 the scaffolding proteins orchestrating this ordered assembly have remained largely unidentified.
Here, we identify and characterize RCC1-2 and APR8 as essential factors directing distinct, sequential phases of daughter cell apical complex construction. Both proteins are recruited with precise spatial and temporal dynamics to the daughter buds, where they function as scaffolds rather than static structural components. APR8 transiently occupies the basal region of the APR specifically in early daughter cells. It is dispensable for conoid and PCR initiation, yet its loss causes APR collapse, abolishes SPMT anchoring, and eventually arrests conoid maturation. In contrast, RCC1-2 localizes beneath the APR basal layer and persists throughout daughter cell development, where it contributes to stabilizing the attachment of SPMTs to the APR. Notably, *in situ* cryo-electron tomography further reveals that the interspersed pillars bridging SPMTs ends to the APR fail to form properly in RCC1-2-depleted parasites. These findings map a hierarchical RCC1-2/APR8-dependent scaffolding process that advances our understanding of parasite replication.

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## Introduction

*Toxoplasma gondii* is a protozoan parasite of significant clinical and veterinary importance. While most human carriers remain asymptomatic, the parasite can be a major threat to immunodeficient individuals. The pathogenesis of this unicellular organism primarily stems from the ability of its tachyzoite form to invade host cells, replicated intracellularly, and eventually egress, leading to cell death and tissue damage. This unicellular eukaryote exhibits a characteristic crescent shape with pronounced apico-basal polarity. Its morphology is maintained by an intricate cytoskeletal scaffold composed of the inner membrane complex (IMC), the underlying alveolin network, and 22 subpellicular microtubules (SPMTs)¹⁻³. These microtubules radiate from a ring-shaped proteinaceous structure, called the apical polar ring (APR), and extend along roughly two-thirds of the parasite's length³,⁴. The APR is further connected to a conical array of spiraling tubulin fibers, known as the conoid, which is capped

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by three preconoidal rings (PCRs)⁵⁻⁸. Within the conoid lie two short microtubules (intraconoidal microtubules, ICMTs) and two distinct classes of secretory organelles: the micronemes and rhoptries¹⁻³,⁸,⁹. Collectively, the APR, PCRs, ICMTs, micronemes and rhoptries form the apical complex. The apical complex operates in tight coordination with the SPMTs and the inner membrane complex (IMC)—a set of double membrane sacs that lie beneath the parasite plasma membrane—to generate the mechanical force necessary for motility, host cell invasion, and egress¹⁰⁻¹⁶.

The tachyzoites exhibit a simplified and rapid division mode called endodyogeny that produce two daughter cells within the mother cell (Fig. 1a). Tachyzoites exhibit a distinctive cell cycle featuring closed mitosis, consisting of three main phases—G1, S, and M—while the G2 phase is thought to be brief and to overlap with the S/M phase¹⁷. During the S and M phases, chromosome replication and organelle duplication occur in coordination with daughter cell development, culminating in cytokinesis that completes daughter cell formation. A single round of DNA replication and mitosis (nuclear cycle) is closely coordinated with the *de novo* formation of the daughter cell scaffold (DCS) in a process referred to as budding or budding cycle³,¹⁸. The bipartite nature of the centrosome enables the independent control of the nuclear (mitosis) and budding cycles¹⁹. It is only recently that the sequence of the DCS formation early events (budding initiation) have been discovered¹⁸,²⁰,²¹. During endodyogeny, centrosome duplication marks the transition from G1 into the S phase and coincides with the beginning of the budding cycle²²,²³. Assembly of the DCS happens in close proximity to the duplicated centrioles, where both apical and basal poles of the daughter cells assemble concurrently²⁰,²¹,²⁴. A striated fiber assemblin (SFA) fiber links the nascent DCS to the centrosome⁴,²⁵, coordinating spatial development of daughter cells. The earliest detectable event in DCS formation is the assembly of the APR which is oriented toward the centrioles and serves as an anchoring site for the cortical microtubules²⁰,²¹. Soon after the nascent APR appears, both the forming conoid and
the ICMTs assemble, accompanied by rafts of 5-6 SPMTs radiating from the APR²¹.
Concurrently, the daughter IMC is assembled and elongates alongside the SPMT cytoskeleton³.
As division progresses, cytoplasmic constriction culminates with the formation of two fully
mature daughter cells, each containing a nucleus and organelles (Fig. 1a). In this process, the
APR plays a pivotal role as the primary site of DCS formation. The APR has been proposed for
many years as a critical anchoring site for the nucleation and organization of cortical
microtubules. However, recent studies suggest that SPMT nucleation may instead originate at
the centrioles where gamma tubulin is localized, with these microtubules subsequently
translocated to the nascent APR²⁰,²⁶. In this context, the nascent APR is best viewed as a critical
anchoring platform that stabilizes not only the SPMTs but also other apical structures, including
the conoid, the ICMTs and IMC, thereby coordinating assembly at the apical pole¹⁸,²¹. The
characterization of the structure and composition of the mature (mother) APR revealed three
distinct concentric layers—upper, middle and bottom layers—each with a distinctive protein
composition⁷,¹⁶,²⁷. Whether these layers assemble in parallel or follow a defined temporal
sequence during DCS formation remains unknown. Notably, despite this architectural
complexity, none of the identified APR proteins have yet been found individually essential for
DCS formation, indicating that key scaffolding components required to organize daughter bud
remain to be discovered.

Here, we describe two critical scaffolding proteins, RCC1-2 and APR8, that govern
distinct stages of DCS budding. APR8 is a bona fide component of the bottom APR layer. It is
required for APR integrity and anchoring of SPMTs to APR, and is necessary to form the APR
and conoid. RCC1-2 localizes just beneath the bottom layer of APR, defining likely a fourth
layer of this complex structure, where it performs a more specific yet crucial scaffolding role
by facilitating the proper anchoring and stabilization of SPMTs to the APR.

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RESULTS

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TgRCC1-2 is recruited at the apical end of daughter cells throughout endodyogeny

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The Regulator of Chromosome Condensation 1 (RCC1) domain is a versatile module involved in the regulation of various cellular processes, including cell division²⁸. In *Toxoplasma gondii* and other apicomplexans, RCC1-containing proteins remain largely uncharacterized, with the notable exceptions of Nd6²⁹, MyoH³⁰, MyoL³¹ and RCC1³², which have been implicated in rhoptry discharge, the PCR-conoid connection and motility, and nucleocytoplasmic transport and cell division, respectively. According to INTERPRO (IPR000408), 15 proteins are predicted to harbor an RCC1 domain in *Toxoplasma* (Supplementary Table 1). To investigate their potential roles, we tagged six of these proteins with a mini auxin-inducible degron (mAID) and a triple HA epitope, allowing conditional protein depletion upon addition of indole-3-acetic acid (IAA) and subcellular localization by immunofluorescence, respectively (Supplementary Fig. 1a–c). These candidates were selected based on their negative fitness scores³³ and predominant expression during the S/M phase³⁴, suggesting potential involvement in the formation of the apical complex and the regulation of its associated components.

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We found two proteins, TGGT1_280770 (RCC1-2) and TGGT1_209130 (RCC1-6), present at the apical end of daughter cells, while the others were mostly diffuse in the cytoplasm of both non-dividing and dividing parasites (Supplementary Fig. 2a). Conditional downregulation of the proteins was achieved by addition of IAA to the culture medium. IFA and western blot analysis confirmed full depletion of proteins after 24 h of IAA treatment (Supplementary Fig. 2b, c). Only depletion of RCC1-2 affects the parasite lytic cycle as shown by complete absence of lysis plaques in monolayers of human foreskin fibroblasts (HFF) infected with RCC1-2-mAID strain after 7 days of auxin treatment (Fig. 1b, c; Supplementary
Fig. 2c, d). RCC1-2 localization at the apical end of daughter cells and its requirement for sustaining the lytic cycle, strongly suggest that it might play an essential role during *Toxoplasma* division, prompting an in-depth characterization of the protein. RCC1-2 is a large protein of 215 kDa, containing three tandem RCC1 repeats in its N-terminal region and two coiled-coil domains in its C-terminal part (Fig. 1d; Supplementary Fig. 1a).

We first investigated the timing of RCC1-2 expression throughout the tachyzoite division process, using nuclear staining alongside two markers of IMC formation—ISP1 and IMC1—which track the formation of the apical cap and middle layer of daughter IMC, respectively. ISP1 is a well-established marker of the apical cap³⁵—a distinctive, cone-shaped plate that forms the uppermost region of the IMC—which is physically connected to the APR. IMC1 staining was performed using specific anti-IMC1 antibodies, while for ISP1, the protein was C-terminally Ty-tagged in the RCC1-2-mAID-HA strain (Supplementary Fig. 3a, b) and detected using anti-Ty antibodies (Supplementary Fig. 3c). In non-dividing parasites, RCC1-2 signal is faint and appears highly diffuse in the cytoplasm (Fig. 1e). As parasites undergo division, two distinct puncta of RCC1-2 emerged in the cytoplasm of dividing cells in proximity to nucleus (Fig. 1e). This expression pattern was consistent with the S/M phase-specific expression profile of RCC1-2, according to ToxoDB data³⁶. We found that RCC1-2 appears prior to the initiation of daughter IMC budding, as shown by the detection of the protein before the incorporation of ISP1 and IMC1 into nascent daughter cells (Fig. 1e, f). As the apical cap of the IMC begins to assemble, RCC1-2 localizes above the ISP1 signal (Fig. 1e) and remains confined at the apical pole of daughter cells until the complete daughter bud maturation; and eventually disappears from individualized newly daughter cells (Fig. 1e, f).

RCC1-2 is recruited after centriole duplication but before SPMTs and conoid formation
We next performed ultrastructure expansion microscopy (U-ExM) to precisely determine the timing and subcellular localization of RCC1-2 in early developmental stages of DCS formation.

To examine RCC1-2's recruitment timing relative to key cytoskeletal structures, we stained α/β-tubulins which allows to visualize the mitotic spindle, the centrioles, the conoid and SPMTs. U-ExM analysis showed that RCC1-2 appears just after centriole duplication (Fig. 1g), a result confirmed by co-staining with centrin 1 marker (Fig. 1h). Initially, the SPMTs appear as discrete 'rafts'—bundles composed of two to four individual SPMTs—positioned facing the centrioles during the early stage of DCS. RCC1-2 signal was systematically detected before the formation of microtubule rafts and conoid structure (Fig. 1g, initiation), suggesting its recruitment to the nascent DCS at a defined early stage of daughter cell biogenesis. At this early stage, RCC1-2 is detected as an arc near the centrioles—the opening of the arc facing the centriole region (Fig. 1g), reminiscent of the nascent APR²¹. Next, it adopted a ring-shaped structure, matching the size of the primordial conoid and is surrounded by the SPMT raft (yellow arrowheads) (Fig. 1g, right panel). The transition from arc-shaped signal to a continuous ring coincides with the emergence of the conoid and the formation of the SPMT array. Notably, this ring-shaped structure consistently forms before the complete nucleation of all 22 cortical microtubules and is maintained until cytokinesis and individualization of daughter cells. RCC1-2 eventually disappears following cytokinesis. RCC1-2 is subsequently lost once cytokinesis is complete. We conclude that RCC1-2 is a daughter-specific protein recruited to the DCS at a very earliest stage of daughter bud initiation and until the end of the endodyogeny process.

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RCC1-2 localizes below the bottom layer APR

To precisely localize RCC1-2 within the apical complex, we first endogenously C-terminally Ty-tagged the conoid protein hub 1 (CPH1)¹¹ (Fig. 2a, Supplementary Fig. 3). During daughter
bud, RCC1-2 localizes as a ring encircling the elongated conoid, approximately midway along its length (Fig. 2b, right panel). It should be noted that in intracellular parasites, the conoid is always retracted and the APR is not located at the conoid base, as observed when conoid is extruded in extracellular parasites. Given that RCC1-2 is exclusively expressed in daughter cells, it is challenging to precisely define the location of RCC1-2 when the conoid is extruded.
To overcome this limitation, intracellular tachyzoites were extracted from infected host cells and subsequently treated with the calcium ionophore A23187 to induce conoid extrusion³⁷.
RCC1-2 signal was detected only in a few rare parasites. In these parasites, RCC1-2 localized beneath the extruded conoid (Fig. 2c), consistent with the localization pattern observed for APR proteins. Additionally, RCC1-2 appears closer to SPMTs than conoid base (Fig. 2c).

The APR itself is a fixed multilayered structure⁷,⁸,¹⁶,²⁷,³⁸, including protein rings such as apical polar ring APR3 (upper layer), APR2 (middle layer), APR1, Kinesin A (KinA) and APR7 (bottom layer), each localizing to specific subregions and conferring stability to the entire complex (Fig. 2a). APR7 extends beyond the APR staining reaching into the spaces between adjacent SPMTs, consistent with a localization at the interface between the APR and the minus ends of the SPMTs¹⁶. Because of the proximity of RCC1-2 with the SPMTs (Fig. 2c), we performed colocalization with the bottom layer proteins KinA and APR7 (description of endogenously C-terminally Ty-tagged lines in Supplementary Fig. 3). We first looked at early stage of division. As anticipated, the recruitment of RCC1-2 to the apical complex precedes that of KinA, which is known to be recruited following the formation of SPMT rafts¹⁰ (Supplementary Fig. 3d). The timing of APR7 recruitment to the DCS had not been investigated before. Here, we demonstrated that APR7 is recruited subsequent to RCC1-2, when the SPMT rafts are formed (Supplementary Fig. 3d). Remarkably, colocalization studies at later stages showed that RCC1-2 localizes beneath both KinA and APR7 (Fig. 2d, e), strongly supporting
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that RCC1-2 occupies a previously uncharacterized space below the bottom layer of APR
defined by APR7.

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Conditional depletion of RCC1-2 causes structural defects of the pellicule

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To elucidate the underlying cause of lethality in absence of RCC1-2, we first examined the
morphology of RCC1-2-depleted parasites. We stained the parasites with markers of the
pellicule and the glideosome: the IMC marker IMC1, the plasma membrane protein SAG1, and
the glideosome component GAP45 (Fig. 3a). In RCC1-2-depleted parasites (24 hours IAA), all
three markers displayed irregular and discontinuous staining patterns, in contrast to the uniform
labelling observed in untreated parasites that maintain their characteristic morphology,
indicating a strong impact on the organisation of the IMC and pellicule. In addition,
multinucleated parasites were observed, indicating a failure in proper daughter cell formation.

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Notably, depletion of RCC1-2 disrupted the typical rosette organization of tachyzoites within
parasitophorous vacuoles (Fig. 3a, b, Supplementary Movies 1 and 2). Labelling of the IMC
with IMC1 and ISP1 further revealed that parasites were at different stages of budding (Fig. 3a),
indicating loss of synchrony during endodyogeny. This asynchrony was consistently observed
in nearly all vacuoles (Fig. 3c). Staining of the apical secretory organelles, marked by AMA1
for micronemes and ARO for rhoptries, showed that these organelles remained associated with
the parasite apex without major differences with wild-type (Supplementary Fig. 4a, b).

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To further investigate the consequences of TgRCC1-2 depletion, we analyzed the
ultrastructure of RCC1-2-depleted tachyzoites using transmission electron microscopy (TEM).
TEM of RCC1-2-mAID-HA parasites treated with IAA for 24h confirmed that the secretory
organelles were correctly positioned to the apical end and that the conoid was readily
identifiable in both the daughter and mother cells (Fig. 3d). The IMC of daughter cells appeared
well positioned relative to the conoid however, on many occasions, it appeared interrupted

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(Fig. 3d). Interruption of the IMC of the mother was also observed (Supplementary Fig. 4c).

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Together, these observations demonstrate that TgRCC1-2 is essential for proper pellicle
organization and the coordinated progression of parasite division.

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RCC1-2 is required for proper anchoring of SPMTs to APR

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Considering that the organization of the IMC relies on the underlying SPMTs and that RCC1-2 is present at the bottom part of the APR, where SPMTs are anchored, we tested whether the global SPMT architecture was also perturbed upon RCC1-2 depletion. Intracellular RCC1-2-mAID-HA parasites treated for 24 h with IAA were processed by U-ExM and SPMTs were stained with anti-α/β-tubulin antibodies. In RCC1-2-depleted parasites, SPMTs were still formed, showing that RCC1-2 does not play a role in tubulin nucleation; however, the symmetry and organization of SPMTs in mother cells was disrupted (Fig. 4a). Parasites lacked the full complement of 22 SPMTs (Fig. 4b; Supplementary Fig. 5a, Supplementary Movies 3 and 4). Three-dimensional reconstruction of extracellular parasites and quantification of SPMTs showed an average of 12 per parasite in absence of RCC1-2 (Fig. 4b, c; Supplementary Fig. 5a), suggesting improper anchoring of SPMTs to the APR. To support this hypothesis, we performed sodium deoxycholate (DOC) extraction on freshly egressed parasites, followed by immunofluorescence to visualize the microtubules. In this experiment, both plasma membrane and IMC membranes are removed by detergent extraction, while the IMC network and the underlying cortical microtubules remain intact and anchored to the conoid via the APR. In absence of IAA, RCC1-2 remained associated with the SPMTs in DOC-extracted parasites (Fig. 4d), showing that RCC1-2 is associated with cytoskeleton structure. In contrast, we found a complete disassembly of the cytoskeleton in RCC1-2-depleted parasites, with only individual microtubules visible by IFA (Fig. 4d), indicating that RCC1-2 plays a crucial role in proper anchoring SPMTs to apical complex. We next examined the impact of RCC1-2 depletion on

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249 the SPMTs of developing daughter cells. In the absence of RCC1-2, the shape of the SPMTs
250 was noticeably altered in daughter buds (Fig. 4e, Supplementary Movies 5 and 6). MORN1,
251 which localizes to the basal end of daughter cells³⁹, is well positioned in the mutant
252 (Supplementary Fig. 6a, Supplementary Movies 5 and 6), but its ring structure was often
253 irregular or appeared as discontinuous segments (Supplementary Fig. 6b). TEM analysis
254 showed that the basal complex retained its typical architecture, defined by an electron-dense
255 ring at the basal end of forming daughters (Supplementary Fig. 6c), supporting that the
256 mishaped basal complex ring structure upon RCC1-2 depletion likely results from disrupted
257 structural support provided by the IMC and SPMTs.

258 Together, these observations indicate that RCC1-2 is crucial for maintaining the proper
259 architecture of SPMTs in developing daughter cells. Because RCC1-2 is present only in
260 daughter cells and absent from mature parasites, these findings support its role as a scaffolding
261 protein that recruits factors required to preserve SPMTs anchoring to the APR in mature
262 parasites. As expected, the associated morphological defects markedly impaired parasite
263 gliding motility (Supplementary Fig. 5c) and host cell invasion (Supplementary Fig. 5d).
264 Nevertheless, although RCC1-2-depleted parasites were able to lyse the parasitophorous
265 vacuole (Supplementary Fig. 5e), their severe motility defect prevented them from spreading to
266 adjacent host cells, as shown by the egress assays (Supplementary Fig. 5f).

267 We next examined the impact of RCC1-2 depletion on the conoid and APR architectures.
268 U-ExM and IFA analyses were performed on RCC1-2-mAID-HA₃ parasites expressing Ty-
269 tagged CPH1, KinA, or APR7, in absence or presence of IAA for 24 hours. In RCC1-2-depleted
270 parasites, the conoid marker CPH1 and the APR markers KinA and APR7 consistently
271 remained localized apically in daughter cells (Fig. 4f–h). The conoid ring structures remained
272 largely intact following treatment, as visualized by fluorescence microscopy (Fig. 4e) and
273 confirmed by TEM (Fig. 3d). KinA staining also appeared preserved overall (Fig. 4g).
However, although APR7 was still detected in daughter cells following 24 hours of IAA treatment, its ring structure appeared interrupted in approximately 71% of vacuoles (Fig. 4h), a phenotype similarly observed in mother cells (Supplementary Fig. 5b). Both KinA and APR7 localize to the bottom layer of the APR, yet APR7 extends vertically beyond this layer, likely reaching the interspace between the SPMTs¹⁶. The severe disruption of the APR7 ring in the absence of RCC1-2, combined with the positioning of RCC1-2 immediately beneath APR7 and the pronounced defect in SPMTs anchoring to the apical complex, supports a role for RCC1-2 in organizing the linkage between SPMTs and the APR rather than structuring the APR itself.

To investigate the ultrastructural basis of these defects, RCC1-2 depleted parasites were subjected to cryo-electron tomography (cryo-ET). Following RCC1-2 depletion, the IMC-APR attachment remained morphologically indistinguishable from wild-type (Fig. 4i, i', j, j'). We also observed focal breakage and loss of structural continuity along the IMC in parasites with intact PM (Supplementary Fig. 4d). Tomogram slices of the apical end showed an overall preserved APR in the mutant: the top, middle and lower layers of the APR retained their band-like appearance in both wild-type (Fig. 4k, k') and RCC1-2-depleted parasites (Fig. 4l, l'). Remarkably, while wild-type parasites showed organized anchoring of the SPMTs to the APR (Fig. 4m, m'), depletion of RCC1-2 resulted in dissociation of some SPMTs from the APR (Fig. 4n, n'), corroborating the results observed by U-ExM. Interestingly, the basal projections sandwiched between adjacent SPMTs and connected to the bottom APR layer (named amorphous APR-associated density [AAD]⁷,²⁷ or interspersed pillars⁴⁰) seemingly exhibit variable disruptions (Fig. 4n', yellow arrows), with the ones next to detached SPMTs showing the most reduced densities (Fig. 4n', red arrows). Measurements of interspersed pillars were obtained from two wild-type and two RCC1-2-depleted parasites. The mean of interspersed pillars length in wild-type parasites was 70 nm (N=15), whereas RCC1-2-depleted parasites exhibited a reduced mean length of 51 nm (N=16). At sites of microtubule disruption, the
average of interspersed pillars length on either side of the broken microtubules was further shortened to approximately 30 nm (N=4). This basal projections, attached to one side of the end of the SPMTs<sup>27</sup> are proposed to function as a linker for the SPMTs, APR and IMC. Together with the abnormal shape of APR7 ring and improper SPMTs anchoring, these results support a role of RCC1-2 in shaping the connection between SPMTs and APR.

## RCC1-2 proximity labeling reveals new daughter-specific proteins

RCC1-2 is associated with cytoskeletal structures and largely insoluble (Fig. 4d, Supplementary Fig. 7c), which prevents the identification of potential direct partners by immunopurification. Instead, to discover new proteins that, like RCC1-2, function at early stages of the budding process, we employed the TurboID biotin ligase proximity labeling approach<sup>41</sup>. A triple HA-tagged TurboID biotin ligase construct was fused to the endogenous C-terminal locus of RCC1-2 (Supplementary Fig. 7a, b). To first assess whether RCC1-2-HA3-TurboID is enzymatically active, intracellular parasites were incubated with or without biotin for 2 hours and IFAs were performed using fluorophore-conjugated streptavidin to label biotinylated proteins and anti-HA antibodies to localize RCC1-2. In the absence of biotin, streptavidin labelling was restricted to the apicoplast, which contains endogenous biotin (Fig. 5a). Upon biotin supplementation, however, an additional streptavidin signal colocalized with RCC1-2-HA3-TurboID at the DCS (Fig. 5a), confirming the enzymatic activity of RCC1-2-HA3-TurboID. These results provided the basis for subsequent immunopurification and identification by mass spectrometry of proteins located in close proximity to RCC1-2. Intracellular parasites were treated with a DOC-based cytoskeletal lysis buffer and biotinylated proteins were captured by immunoprecipitation using streptavidin-coated magnetic beads followed by mass spectrometry analysis. Western blot analysis showed a marked increase in biotinylated proteins in presence of biotin (Fig. 5b, Supplementary Fig. 7c). A total of 460 proteins were identified
with a marked enrichment of proteins associated with the apical, and basal complexes (Supplementary Table 2). The identified apical complex proteins include components of the APR such as APR1, RNG2 and KinA<sup>10,42</sup>. Most of known basal complex proteins were enriched (BCC0, BCC2, BCC3, BCC4, BCC5, BCC6, BCC7, BCC10, BCC11, FIKK, MyoJ<sup>24,43-45</sup>). Among enriched proteins are also α and β tubulins, many proteins of the γ-tubulin complex (GCP2, GCP3, GCP4 and GCP5)<sup>20,26,36</sup>, but also many apical cap proteins (AC2, AC7, AC8, AC10, AC12<sup>12,36,46,47</sup>) and some IMC proteins such as IMC29<sup>48</sup>, IMC30<sup>48</sup>, IMC33<sup>47</sup> and IMC43<sup>49</sup> that are recruited at the early bud during endodyogeny. This comprehensive proteomic profiling aligned well with the early location of RCC1-2 to DCS and with previous studies showing coordinated development of the IMC, SPMTs, apical and basal complexes during daughter cell assembly in *Toxoplasma*.

To select the most relevant RCC1-2-proximal candidates associated with DCS for characterization, we applied selection criteria based incorporating their enrichment in the TurboID assay, low fitness-conferring scores indicating essentiality<sup>33</sup> and cyclical expression profiles peaking in the S/M phase of the cell cycle<sup>34</sup>. Furthermore, to specifically seek for proteins transiently expressed at the daughter bud, we prioritized candidates with uncharacterized localization patterns in the hyperLOPIT dataset<sup>50</sup>. Transiently expressed proteins during endodyogeny are indeed usually not found in the spacial proteomic analysis, as exemplified for RCC1-2. This combined approach enabled us to refine the list to proteins most likely involved in early DCS assembly and function. Among the 11 uncharacterized proteins thus filtered (Supplementary Table 3), we independently characterized two in a separate study—TGGT1_293170 and TGGT1_255700—and showed that both are associated with a fiber structure that colocalises with SFA (unpublished, manuscript in preparation) and play a role in daughter cell formation, thereby validating our approach. Bioinformatics analysis using INTERPRO<sup>51</sup> and HHPRED<sup>52</sup> detected a tumor overexpression gene (TOG) domain for
349 TGGT1_312590 and a Tubulin Tyrosine ligase (TTL) domain for TGGT1_244500, which is
350 predicted to be a tubulin polyglutamylase (Fig 5c). Notably, both TOG and TTL domains
351 exhibit functional links to tubulin by interacting directly with tubulin and playing critical roles
352 in regulating microtubule dynamics<sup>53,54</sup>. Interestingly, like RCC1-2, many of these proteins
353 contain a coiled-coil segment (Supplementary Table 3).

354 We next investigated the localization and function of TGGT1_212780,
355 TGGT1_312590 (TOG-containing protein) and TGGT1_244500 (Tubulin Tyrosin ligase) by
356 endogenously tagged them with the mini auxin-induced degron (mAID) and a triple HA tag
357 (Supplementary Fig. 7d). IFA revealed that TGGT1_312590 exhibited faint diffuse
358 cytoplasmic distribution in both non-dividing and dividing parasites, while the expression of
359 the other two proteins was particularly enriched to dividing parasites (Fig. 5d). The Tubulin
360 Tyrosin ligase TGGT1_244500 was recruited to the daughter IMC (Fig. 5d). TGGT1_212780
361 displayed a localization pattern characterized by two puncta in parasites initiating division,
362 similar to RCC1-2 and few APR proteins (Fig. 5d). Next, we assessed the ability of the mutants
363 to complete the lytic cycle upon depletion of the proteins by adding IAA in the culture media.
364 Depletion of the proteins was efficient for all transgenic lines, as shown by western blot (Fig.
365 5e) and IFA (Supplementary Fig. 7e) analysis after 24 h of auxin treatment. No significant
366 reduction in plaque area was observed for the Tubulin Tyrosin ligase and TGGT1_312590
367 mutants (Fig. 5e, f) in monolayers of HFF cells, after 7 days of auxin treatment. In contrast
368 TGGT1_212780-mAID failed to form lysis plaques (Fig. 5e, f). Based on its localization and
369 essential role, we thus further characterized TGGT1_212780.

370

371 **TGGT1_212780 associated with the bottom layer of APR, and named APR8**

372 TGGT1_212780 can be detected as soon as parasites initiate division, presenting as two distinct
373 puncta near the nucleus prior to the formation of cytoskeleton structures (Fig. 6a,
Supplementary Fig. 8a), subsequently localizing to the apical end of DCS (Fig. 6b). Notably, TGGT1_212780 signal disappears very early, as the daughter IMC begins to elongate (Fig. 6b; Supplementary Fig. 8a), appearing to be much more transiently present in daughter cells compared to RCC1-2, which remained until cytokinesis (Fig. 1e, 1f). As observed for RCC1-2, TGGT1_212780 initially localizes as an arc positioned above the newly duplicated centrosomes (Fig. 6a), and then transitions into a complete ring structure at the apical pole of daughter cells (Fig 6b).

In order to precisely determine the localization of TGGT1_212780 within the apical complex, we co-labeled TGGT1_212780 with: i) preconoidal marker (PCR1)¹⁴, ii) conoid marker CPH1¹¹, iii) APR markers (upper layer APR3; middle layer APR2; bottom layer KinA and APR7)¹⁶, iv) striated fiber SFA2²⁵, and v) the newly described RCC1-2 protein. All the proteins were endogenously tagged with either a Ty or a Myc tag (Supplementary Fig. 8b, c). Examining both side-view (Fig. 6c) and top-view (Supplementary Fig. 8d) images of the colocalization of TGGT1_212780 with CPH1 and PCR1 showed that TGGT1_212780 localized beneath the PCRs but above the conoid, consistent with an APR localization. Colocalization analysis with the distinct APR layers revealed that TGGT1_212780 localized at a lower level than APR3 and APR2, while showing complete colocalization with KinA and partial overlap with APR7 at the bottom layer (Fig. 6c, Supplementary Fig. 8e). TGGT1_212780 was clearly localized above RCC1-2 (Fig. 6c, Supplementary Fig. 8e) and encircled the SFA2-labeled striated fiber, which connect the apical complex to the centrosome in daughter cells (Supplementary Fig. 8f). Based on this specific localization pattern, we concluded that TGGT1_212780 is a daughter APR protein associated with the bottom layer of the APR. According to the current annotation of APR proteins, TGGT1_212780 was hereafter designed APR8.

398
399 **Layered organization of the APR is stablished early during DCS**

400 The multilayered organization of the APR has been characterized in mature parasites, but the
401 timing of its assembly during DCS has not yet been investigated. Our colocalization and U-
402 ExM analyses focusing on daughter cells provided the opportunity to address this question. Co-
403 localization revealed that APR8 is incorporated earlier than KinA and APR7, while its
404 characteristic arc-shaped signal appears concomitantly with APR2 (middle APR layer), APR3
405 (upper APR layer), and the PCR marker PCR1 (Fig. 6d). At early stage of endodyogeny, the
406 signals for APR2, APR3, and PCR1 were already clearly resolved as distinct structures from
407 APR8. Moreover, APR8 appears positioned below both APR2 (middle layer) and APR3 (upper
408 layer), consistent with its localization in the bottom layer of the APR and supporting the idea
409 that the three layers are established very early during DCS, before the emergence of cytoskeletal
410 structures. In addition, APR8 and RCC1-2 were detected at the same time in the DCS, with the
411 APR8 arc positioned above the RCC1-2 signal (Fig. 6d). Together, these observations indicate
412 that PCR and APR assemblies are established concomitantly and that their respective layers are
413 already segregated at the onset of bud initiation, with APR8 representing an early marker of the
414 basal APR layer.

415

416 **APR8 acts as a scaffold for APR organization and subsequent proper apical complex**
417 **development**

418 To dissect the role of APR8 in early steps of the DCS formation, parasites were allowed to
419 replicate for 24 hours and either left untreated or treated with IAA for 6 hours which represent
420 the time needed to complete their cell cycle. Complete depletion of APR8 was observed after 4
421 hours of treatment (Fig. 7a). After 6 hours of incubation with IAA, only the SPMTs of the
422 daughter cells are affected; 98% of daughter cells showing a severely disorganized cytoskeleton
423 by U-ExM (Fig. 7b). At 24h, nearly all vacuoles exhibiting a distorted IMC architecture
adopting a swollen shape (Fig. 7c). TEM of intracellular APR8-mAID-HA parasites incubated
for 6 hours with IAA confirmed the important perturbation of the daughter cell IMC, that
appears as discontinuous structure flotting in the cytoplasm of the mother cell (Fig. 7d, e). These
finding showed that assembly of both the daughter IMC and SPMTs are not properly performed
in absence of APR8.

We next quantified the proportion of tachyzoites expressing various apical complex
markers after depleting APR8 for 6 hours with IAA. As expected at this time point, the depletion
did not affect the apical structures of the mother (PCR, conoid and APR) (Fig. 7e;
Supplementary Fig. 9a). For quantification in daughter cells, we measured signal presence in
parasites exhibiting elongated SPMTs to avoid bias from markers not expressed at early stages
of the budding process, such as CPH1 or even later markers KinA and APR7. The recruitment
of all analyzed markers (CPH1, PCR1, APR3, APR2, KinA, and APR7) was affected to varying
degrees (Fig. 8a, b). Approximately 20% and 30% of parasites failed to recruit the conoid
(CPH1) and PCR (PCR1) markers, respectively. The defect reached 60% for the upper APR
layer (APR3) and 70% for the middle APR layer (APR2), whereas the most pronounced
disruption occurred in the bottom APR layer, with nearly all daughter cells lacked KinA and
APR7. The presence of RCC1-2 in daughter cells was only slightly affected upon APR8
depletion (20%). The overall protein levels remained unchanged upon auxin treatment,
demonstrating that the defects observed were not due to degradation of the inspected proteins
(Supplementary Fig. 8c).

While the conoid marker CPH1 was discernible, the classical ring shape structure was
never observed upon removal of APR8 (Fig. 8b-e; Supplementary Fig. 9d). Instead, the conoid
adopted an abberant ‘arc’ shape, indicating that APR8 also plays a role in proper conoid
maturation. TEM analysis further revealed nascent conoid in daughter cells, and that the IMC
failed to align with the apical edge of the conoid, in contrast to wild-type parasites (Fig. 7e),
449 illustrating improper assembly of daughter cell material. Notably, nascent conoid was still
450 connected to centrioles via the SFA fiber in TEM analysis (Supplementary Fig. 9e), a result
451 confirmed by tagging the SFA2 protein in APR8-mAID line and counting the number of
452 parasites with SFA labeling by IFA (Supplementary Fig. 9f-h). This result indicates that
453 division initiation and polarity establishment by the SFA fiber proceeded unperturbed in the
454 absence of APR8. PCR1 staining appeared largely unaffected (Fig. 8b, c); however, its small
455 size in daughter cells³¹ precluded definitive conclusions on its shape. PCR1 remained associated
456 with one extremity of the arch formed by CPH1 in APR8-depleted parasites, as shown by
457 costaining of the two proteins in APR8-mAID parasites (Fig. 8c) suggesting that APR8 is not
458 associated in the recruitment of PCR1 and CPH1 to the PCRs and coinoid, respectively. In the
459 absence of APR8, the upper and middle markers of APR—though sometimes still visible—lost
460 their characteristic ring-shaped pattern (Fig. 8b). Both are visible close to conoid, but never
461 formed a ring structure (Fig. 8d, e). Moreover, the daughter SPMTs sometimes radiate from the
462 APR2 or APR3 signal. The transition of the arc to ring-like arrangement of RCC1-2 was also
463 profoundly disrupted upon APR8 depletion (Fig. 8b). In contrast, tagging APR8 in RCC1-2-
464 mAID strain (Supplementary Fig. 8b, c) showed that its normal localization and shape were
465 largely preserved upon RCC1-2 depletion (Fig. 8f). This asymmetric dependency indicates that
466 RCC1-2 functions downstream or independently of APR8 in apical complex assembly.
467
468 Together, these observations indicate a complete defect in multi-layered APR formation,
469 despite recruitment of early markers APR3 and APR2 to nascent buds. This likely impairs
470 subsequent recruitment of late APR markers KinA and APR7 and compromises proper SPMTs
471 anchoring. In contrast, APR8 is dispensable for PCR and conoid initiation but essential for
472 maturation and conoid assembly, establishing APR8 as a critical scaffold for coordinated
473 daughter cell morphogenesis (Figure 8g).

473
474 DISCUSSION

475 *T. gondii* tachyzoite endodyogeny is regulated at transcriptional and post-translational levels¹⁸.

476 Recent methodological advances, such as U-ExM have shed light on DSC events²⁰,⁵⁵,

477 highlighting the APR's central role in DCS construction—yet no essential proteins for its

478 formation had been identified until now. In this study, we describe two essential scaffolding
479 proteins for DSC assembly: RCC1-2, which participates in anchoring SPMTs to the APR; and

480 APR8, which ensures the correct assembly and maintenance of the multi-layered APR
481 architecture. Both proteins are transiently expressed during parasite cell cycle: RCC1-2 persists

482 until daughter cell maturation is complete, while APR8 disappears earlier, before daughter cell
483 elongation. These dynamics indicate a scaffolding rather than permanent structural roles.

484 Although both APR8 and RCC1-2 arrive early at the DSC construction site, their distinct
485 localizations seem to be the determining factor for their specific role during daughter cell

486 formation. APR8, a resident of the bottom layer APR, controls the overall assembly of the APR
487 architecture: in its absence, most APR markers fail to associate with the nascent daughter bud

488 or, when present, no longer display the typical ring-like organization. In APR8-depleted
489 parasites, SPMTs are synthetized but are not always attached to a specific structure, reinforcing

490 the role of the APR as the main anchor for the SPMT⁵⁶, rather than a site for their nucleation as
491 long proposed. RCC1-2, positioned beneath the basal APR layer, is required for robust SPMT

492 attachment—a defect reminiscent of that observed in APR1/KinA double mutants¹⁰, both
493 components of the basal APR. Interestingly, Leung et al. reported that the detachment of the

494 SPMTs in the ΔKinAΔAPR1 parasites occur at later daughter development¹⁰. It is worth to
495 mention that some bottom APR proteins, like RNG1⁵⁷, APR1⁵⁵ or APR7 (this study) are
496 recruited late in the buding process. These observations suggest that the APR/SPMTs

497 connection needs to be continously strenghtened through the recruitment of late components⁵⁵.

498 RCC1-2, which remains associated with the DSC until the completion of daughter maturation,
RCC1-2, which remains associated with the DSC until the completion of daughter maturation, is an ideal scaffolding protein to sustain this function throughout DSC assembly. In contrast to APR8, RCC1-2 depletion does not grossly perturb APR architecture, as shown by APR-layer markers and cryo-ET, with APR7 being the only marker that loses its typical ring-like distribution. APR7 spans the interface between the APR and the apical ends of SPMTs, although its precise position remains unresolved. Consistently, RCC1-2 sits just beneath APR7¹⁶, and cryo-ET of RCC1-2-depleted parasites reveals SPMTs detachment and shortened interspersed pillars. Together, these observations demonstrate that RCC1-2 is required for proper interspersed pillars assembly and establish these newly described elements as critical anchors for SPMTs at the APR. Notably, the IMC remains correctly attached to the APR in RCC1-2-depleted parasites, indicating that APR-IMC linkers are intact. Since interspersed pillars have been proposed to occupy the expanded region below the amorphous AAD ring that links the APR to the IMC, our data point to a narrow function for RCC1-2 in building the interspersed pillars themselves, and that other scaffolding proteins likely contribute to connect the IMC to the AAD ring. As a transient DCS component, RCC1-2 is well placed to recruit yet-unknown pillar constituents.

In addition to defective SPMTs anchorage, RCC1-2 depletion causes interruptions in the IMC, which could arise from mechanical stress during cytoskeletal elongation when SPMTs detach from the APR. This detachment may also contribute to altered basal complex morphology observed. However, because apical and basal poles form concomitantly early during DCS assembly ²¹,²⁴, RCC1-2 might also directly influence IMC organization, including plate sutures and basal pole development, independently of its role in SPMT anchoring.

In this study, we demonstrate that the four APR layers assemble concomitantly during nascent APR formation—with their distinct localizations already established prior to conoid assembly—suggesting that this hierarchy is established at the onset of their insertion into the
nascent APR. We further detect PCR1, a PCR marker¹⁴, at the same early DCS stage, and similar early recruitment is seen for Centrin2, which also localizes to the PCR⁵⁵. Thus, APR and PCR jointly pioneer apical pole construction ahead of cytoskeletal structures such as the conoid or ICMTs. To date, SFA fiber nucleation is the only event known to precede nascent APR appearance⁴,²⁵. The absence of perturbation of the SFA2 marker in absence of APR8 suggests that the SFA fiber nucleation happens upstream and independently from of the nascent APR formation. In fact, the function of the SFA fiber precedes that of γ-tubulin as shown in an auxin-inducible mutant of SFA2²⁰. SFA fiber has been hypothesized to act as a spatial organizer of the DCS formation site²⁵, but its function needs to be further investigated to understand it role in APR formation.

In parallel, an independent study identified APR8 (named ASAF1) as a crucial factor for daughter cell formation⁵⁸. In our study, we precisely localized this protein to the APR basal layer and adopted the APR-based nomenclature by naming it APR8. Our work confirms most of their findings but also reveals differences attributable to the depletion strategies used. Here, we employed a fast-acting auxin-inducible degron that depletes APR8 within tens of minutes, enabling phenotypic analysis after 4–6 h of depletion, before secondary defects accumulate. By contrast, Li et al.⁵⁸ used the inducible Cre system⁵⁹ that induces genetic excision of the target sequence in a much slower kinetic (24 to 48 hours). The fast degradation of APR8 in our system allowed an evaluation of the phenotype after 4-6 hours of depletion, before the accumulation of indirect phenotypes, and a close inspection of the the apical complex structures formation in absence of APR8. In our system, PCR, conoid, and APR markers are clearly recruited near duplicated centrioles, yet none achieve their normal morphology. The conoid consistently appears as an immature arc reminiscent of the primordial conoid seen by U-ExM⁵⁵, indicating a maturation rather than a recruitment defect. PCR1 remains associated with the conoid, typically at one extremity, but its small size precludes definitive structural assessment. By
contrast, APR2 (middle layer) and APR3 (upper layer) are often undetectable at the apical pole
and, when present, never form rings. Likewise, RCC1-2—marking the deepest
layer/interspersed pillars—is recruited but fails to assemble into a continuous ring, and the late
basal APR markers APR7 and KinA are not detected near the conoid. Together, these data
indicate a profound failure of APR assembly upon APR8 loss, which compromises downstream
events such as anchoring of newly polymerized SPMTs and the IMC, as also seen by TEM.
Although PCRs and the conoid still initiate without APR8, the abnormal conoid shape
underscores that its maturation is coupled to global APR integrity. Recent structural and
molecular work shows that the conoid-APR connection relies on tethering elements formed by
RNG2, which bridges the conoid to the APR in mature parasite<sup>42,60,61</sup>. Notably, RNG2 depletion
does not block conoid formation or maturation but causes its detachment from the apical
complex in mature parasite, suggesting that other APR proteins are crucial for stabilization and
maturation of conoid during endodyogeny. The APR ring may also template the conoid’s
transition into a fully cylindrical structure. The fact that APR8 is dispensable for initial PCR or
conoid formation supports the hypothesis that the PCR—which precedes conoid assembly—
might act as an organizing center for conoid biogenesis. The orientation of conoid fiber minus
ends toward the PCR, with plus ends near the APR, further aligns with a model in which the
PCR organizes conoid assembly. To date, no mutants specifically defective in PCR biogenesis
have been identified to test this hypothesis. Known mutants such as GCP, PCR4, and PCR5<sup>31,58</sup>,
are crucial for maintaining a mature PCR but dispensable for its initial formation, suggesting
that additional, as-yet-unknown factors drive PCR initiation. PCR7, a transiently expressed
PCR component during DCS formation<sup>14</sup>, would be an attractive candidate, but its non-
essentiality argues against a primary scaffolding role. Similarly, depletion of ERK7 induces a
complete loss of the PCR, conoid, and APR, but this effect is observed only in mature parasites.
The core scaffolding proteins that directly organize PCR and conoid architecture therefore remain to be identified⁴⁶,⁴⁷.

Both scaffolding proteins, APR8 and RCC1-2, are specific to coccidian parasites—a subclass of apicomplexans (including *Eimeria* spp.) characterized by an elaborate conoid. This coccidian specificity might reflect parasite-specific features of the APR and its considerable variability among apicomplexans and across developmental stages. In *Toxoplasma*, the APR appears as three distinct density layers from which 22 evenly spaced SPMTs radiate. In *Plasmodium*, APR structure varies markedly by stage⁶²,⁶³: merozoites and sporozoites have a single ring, with 2–3 asymmetrically arranged SPMTs in merozoite, while sporozoites feature 13 closely spaced SPMTs plus one opposing microtubule at tilted angles. Ookinetes exhibit the most complex "apical collar" with two concentric layers and basal tentacles supporting ~50–60 staggered SPMTs. These divergent architectures illustrate how stage- and species-specific APR designs tune cell shape and mechanics.

Overall, our study pionnered the understanding of the molecular players of the initiation of the apical complex construction, specifically the APR and connecting materials with SPMTs of *Toxoplasma gondii*. By defining these early assembly factors and their spatial organization, our work connects the molecular blueprint of APR-SPMTs coupling and links it to the apical complex's essential roles gliding motility, host cell invasion, and egress.

## Materials & Methods

### *Toxoplasma* culture conditions - Antibodies used

*T. gondii* RH tachyzoites (Type I strain) were routinely grown in Human Foreskin Fibroblasts (HFFs, ATCC CRL 1634) in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 5% fetal bovine serum (FBS, Biosera), 2mM glutamine (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C + 5% CO₂. Transgenic lines were generated using
either the RHΔKu80TATi strain, which expresses the TATi transactivator for the TetOff system⁶⁴, or the RHΔKu80TIR1 strain, which expresses the Tir-1 receptor for the auxin inducible degron system⁶⁵ (miniAID). Mycophenolic acid (25 µg/mL) and xanthine (50 µg/mL) were added to the culture media to positively select transformants carrying the hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) drug resistance cassette. For conditional knockdown, 0.5 mM auxin (indole-3-acetic acid, referred to as IAA (Sigma-Aldrich)) was added to the medium.

Antibodies used and their dilution are listed in Supplementary Table 4.

## Generation of *Toxoplasma* tagged and knockdown strains

All parasite strains used in this study were generated by CRISPR/Cas9-mediated homology-directed repair. For C-terminal epitope tagging, 40 µg of the pU6-Cas9-YFP carrying the sgRNA targeting the 3' untranslated region (3'UTR) of the gene of interest (GOI) was co-transfected with a PCR product containing the corresponding homology regions and the desired tagging cassette (mAID-HA-HXGPRT, HA-TurboID-DHFR, Ty-DHFR, or Myc-DHFR). The mAID-HA-HXGPRT cassette was amplified from the plasmid pTUB8YFP-mAID-3⁶⁵. The pU6-sgRNA plasmids were constructed by annealing and cloning the sgRNA primers into the pU6-Cas9-YFP plasmid using the BsaI restriction sites. The list of all tagged lines, primers and RNA guides (gRNAs) used are listed in Supplementary Table 4.

For all constructs, freshly egressed *T. gondii* tachyzoites (20 x 10⁶) were electroporated with approximately 15 µg of the pU6-Cas9-YFP plasmid and 100 µL of PCR-amplified homologous recombination (HR) template using an Electro Cell Manipulator 630 (BTX) with the following settings: 2.02 kV, 50 Ω, and 25 µF⁶⁶. Transfected parasites were allowed to infect HFFs monolayers. After transfection, positive transformants were recovered by drug selection for transformants carrying the hypoxanthine-xanthine-guanine phosphoribosyl transferase
(HXGPRT) drug resistance cassette or by fluorescence-activated cell sorting (FACS) based on
Cas9-YFP fluorescence for C-terminal tagging with HA-Turbo, Myc or Ty epiopes. Clones
were isolated by limiting dilution and screened by PCR for correct integration with Phire™
Tissue Direct PCR master mix (Thermo Scientific) protocol, as previously described⁶⁷. The
primers used to validate the integration are listed in Supplementary Table 4.

628

629 *Toxoplasma* invasion assay

630 HFF cells grown on coverslips were infected with freshly egressed *T. gondii* tachyzoites (5. 10⁶
631 /coverslips) pre-treated for 24h ± IAA. Parasites were incubated on ice for 20 min to
632 synchronize invasion prior to being placed in a 38.5°C water bath for 5 min to allow invasion.
633 Infected cells were fixed in 4% paraformaldehyde (PFA) (w/v) in Hank's Buffer Salt Solution
634 (HBSS; Gibco) for 20 min at room temperature. To distinguish intracellular from extracellular
635 parasites, immunofluorescence assays were performed using a dual-staining antibody as
636 previously described⁶⁸. Non-permeabilized extracellular parasites were incubated first with
637 10% FBS/HBSS blocking solution and then stained using mouse mAbs T41E5 anti-SAG1
638 antibodies⁶⁹ (1:2,000⁶⁶) in 2% FBS/HBSS at 37°C. To visualize intracellular parasites, samples
639 were then permeabilized with 0.1% saponin in PBS for 15 min, incubated in blocking solution
640 for 1 hour and stained using rabbit anti-GAP45 antibodies (1:9,000⁷⁰) with 2% FBS in
641 Phosphate Buffered Saline (PBS). After three washings with PBS, the samples were incubated
642 with AlexaFluor594 goat anti-mouse (1:4,000) and AlexaFluor488 goat anti-rabbit (1:10,000)
643 secondary antibodies (Invitrogen). Extracellular and intracellular parasites were scored in 20
644 fields per coverslips in triplicates with a Leica Thunder microscope, with a 100× oil objective
645 NA = 1.4, equipped with the sCMOS 4.2MP camera, using Leica Application Suite X (LAS X)
646 software (Leica Biosystems). The values were normalized to that of the control line (RHΔΚΥ80
647 Tir1).
648

649

### Toxoplasma plaque assay

650 HFF cells grown in 24-well plates were infected with 7000 of freshly egressed *T. gondii* tachyzoites per well. After a serial dilution of *T. gondii* tachyzoites, parasites were allowed to grow for 7 days with or without IAA at 37°C + 5% CO₂. HFF cells were then fixed with 4% PFA/PBS for 20 min, washed once with PBS prior to be stained with 1% crystal violet (Sigma). Image of the lysis plaques were acquired with Olympus MVX10 macro zoom microscope, equipped with a Zeiss MRM2 Camera. Lysis plaque area was measured using ImageJ software. Graphs are presented as mean values from three independent experiments, including three technical replicates each.

658

659

### Toxoplasma replication assay

660 HFF cells grown on coverslips were infected with 2x10⁵ of freshly egressed *T. gondii* tachyzoites for 2 hours to allow invasion. HFFs cells were then washed five times with HBSS to remove remaining extracellular parasites. Parasites were then allowed to grow with or without IAA at 37°C for 16 hours. Parasites were fixed with 4% PFA/PBS for 20 min, permeabilized with 0.1% Triton X-100 and incubated with 5% BSA/PBS blocking solution. 665 Immunofluorescence assay was performed with rabbit anti-GAP45⁷⁰ (1:9,000) and 666 AlexaFluor488 goat anti-rabbit (1:4,000; Invitrogen) antibodies diluted in 2% BSA/PBS. 667 Nuclei were stained with 16 µM Hoechst before mounting the coverslips onto microscope slides 668 using Immunomount (Calbiochem). The number of vacuoles containing either 1, 2, 4, or 8 669 nuclei per vacuole were scored with a Leica Thunder microscope, with a 100× oil objective 670 NA = 1.4, equipped with the sCMOS 4.2MP camera, using Leica Application Suite X (LAS X) 671 software (Leica Biosystems) and expressed as a percentage. A total of 200 vacuoles/coverslips
672 were scored for each condition. Graphs are presented as mean values from three independent
673 experiments, including three technical replicates each.

674

675 *Toxoplasma egress assay*

676 HFF cells grown on coverslips were infected with 1x10⁵ of freshly egressed *T. gondii*
677 tachyzoites following 30h of infection in presence or absence of IAA. To stimulate egress,
678 infected cells were incubated with calcium ionophore A23187 (3 µM) or dimethyl sulfoxide
679 (DMSO, control) for 8 min at 37°C w and then fixed with 4% PFA/PBS for 20min.
680 Immunofluorescence assay was performed using rabbit anti-GAP45⁷⁰ (1:9,000) and mouse anti-
681 GRA3 hybridoma⁷¹ (1:100) followed by incubation with AlexaFluor594 goat anti-rabbit
682 (1:4,000) and AlexaFluor488 goat anti-mouse (1:4,000) antibodies (Invitrogen). Nuclei were
683 stained with 16 µM Hoechst before mounting the coverslips onto microscope slides using
684 Immunomount (Calbiochem). Egress events were analyzed by assessing the presence of GRA3
685 proteins in intact and ruptured parasitophorous vacuoles stained with GAP45. 200 vacuoles
686 were scored for each condition. Graphs are presented as mean values from three independent
687 experiments, including three technical replicates each.

688

689 **Western blotting of *Toxoplasma* proteins**

690 For western blotting of infected HFF monolayers, parasites were cultured 30h prior to be
691 harvested by scraping and syringing out 3 times with a 25-gauge needle. Parasites were pelleted
692 by centrifugation at 800 x g for 5 min, washed once in PBS, pelleted again and resuspended in
693 Laemmli buffer 4X supplemented with 40 mM dithiothreitol (DTT; Sigma). Proteins extracts
694 were separated on 10% Novex NuPAGE Gels (Invitrogen) and transferred to 0.45 µm PVDF
695 (Immobilon®-P, Millipore) membranes. Blots were blocked either with 5% dried milk or 3%
696 BSA in TNT (15 mM Tris, 140 mM NaCl, and 0.05% Tween 20, pH 8) and incubated with
697 antibodies diluted in blocking solution. Detection was performed using Clarity Max™ Western
698 ECL (Bio-Rad) and imaged with a Chemidoc System (Bio-Rad).

699

700 Sodium deoxycholate extraction

701 Freshly egressed parasites pre-treated for 24h ± IAA were settled onto Poly-L-Lysine coated
702 coverslips by centrifugation at 400 x g for 1 min. Subsequently, parasites were treated with
703 DMSO (as control) or 10 mM of sodium deoxycholate (DOC; ThermoFisher) for 20 min at
704 room temperature. Parasites were then fixed with ice-cold methanol for 8 minutes prior to
705 immunostaining using mouse anti-acetylated αtubulin (Santa Cruz Biotechnology, sc-23950)
706 and rabbit anti-HA (Abcam) antibodies followed by incubation with AlexaFluor488 goat anti-
707 mouse (1:4,000) and AlexaFluor594 goat anti-rabbit (1:4,000) antibodies (Invitrogen).

708

709 Biotinylation by TurboID

710 RCC1-2HA3-TURBOID parasites were cultured in HFFs monolayers for 28 hours in normal media.
711 After a wash with biotin-free medium, parasites were then grown in biotin-free DMEM (as
712 control) or biotin-free DMEM supplemented with 150 mM D-biotin (Sigma-Aldrich) for 2
713 additional hours. Intracellular parasites were harvested by scraping and syringing out 3 times
714 with a 25-gauge needle, washed in PBS and then lysed with a cytoskeletal lysis buffer 1 (50
715 mM Tris [pH7.5], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, Complete
716 Protease Inhibitor Cocktail (Roche)) for 30 min on ice. Lysates were centrifuged for 15 min at
717 14,000 x g at 4°C. Following centrifugation, the pellet corresponding to the cytoskeletal and
718 IMC fraction was lysed using lysis buffer 2 (50 mM Tris [pH7.5], 150 mM NaCl, 1% SDS, 1%
719 Triton X-100, Complete Protease Inhibitor Cocktail (Roche)) for 30 min and then incubated
720 with Steptavidin magnetic beads (ThermoFisher Scientific) overnight at 4°C under agitation.
721 Beads were collected and washed five times with wash buffer (50 mM Tris [pH7.5], 150 mM
NaCl, 0.1% SDS, 1% Triton X-100, Complete Protease Inhibitor Cocktail (Roche)) prior to be
eluted in Laemmli buffer 4X. Supernatants from the eluates were loaded onto a 10% acrylamide
SDS-PAGE gel, transferred to 0.45 µm PVDF (Immobilon®-P, Millipore) membranes. Blots
were blocked with 3% BSA in TNT and incubated with horseradish peroxidase (HRP)-
conjugated Streptavidin (1:5,000; Sigma-Aldrich) antibodies diluted in blocking solution.
Detection was performed using Clarity Max™ Western ECL (Bio-Rad) and imaged with a
Chemidoc System (Bio-Rad).

For IFA, infected monolayers were incubated for 2 h with biotin prior to fixation with
4% PFA for 20 min. Coverslips were then processed according to the standard
immunofluorescence staining protocol described for invasion and replication assays.

For mass spectrometry analysis, electrophoresis was stopped once the samples had
migrated 1-2 cm into the gel. Proteins were stained with colloidal blue (Invitrogen) prior to be
analyzed by mass spectrometry (MSPP platform – INRAE Montpellier).

Mass spectrometry analysis of *Toxoplasma* proteins

Sample preparation

Gel bands were manually cut and re-cut into small pieces (2mm2) that were transferred to
2 mL low binding tubes (Thermo Scientifics). Bands were first washed with 500 µl of water
and then 500 µl of 25 mM NH4HCO3. Destaining was performed twice in the presence of 500
µl of 50% acetonitrile (ACN) in 25 mM NH4HCO3. Gel bands were dehydrated twice by 500
µl of 100% acetonitrile, and finally dried at room temperature. Destaining is followed by the
reduction of disulfide bridges with 500 µl of 10 mM DTT at 56°C for 45 min, then supernatant
was removed and cysteins were alkylated with 500 µl of 55 mM iodoacetamide during 30 min
on a vortex in dark. Gel bands were washed two times with 500 µl of 50% ACN in 25 mM
NH4HCO3, then dehydrated by 500 µl of 100% ACN, and finally dried at room temperature. 30
747 microliters of a trypsin solution (Sequencing Grade Modified Trypsin, Promega, Madison,
748 USA), at a concentration of 0.0125 µg/µl in 25 mM NH4HCO3, was added to every gel region,
749 and sufficient carbonate buffer (25 mM NH4HCO3) was added to cover the gels and were kept
750 for 10 min on ice. A volume of carbonate buffer was added once more to cover the gels before
751 the samples were kept another 10 min at room temperature. Digestion was performed overnight
752 at 37°C then peptides were extracted by addition 100 µl formic acid (FA) 2 %. Digests were
753 first voretexed for 10 minutes and supernatants were transferred into 2 mL low binding tubes.

754 Gel bands were extracted twice by addition of 100µL of 80 % ACN. After solvent evaporation
755 in a vacuum centrifuge, peptides were resuspended in 10 µl 2% FA. Then purified with a micro
756 tip C18 (Zip-Tip- C18 Millipore Corpration Billerica MA, USA). Peptides were eluted with a
757 solution containing 2% FA (v/v) and 80% ACN (v/v) and dried in a vacuum centrifuge until
758 total evaporation. Peptides were resuspended in 8 µl 2% FA before LC-MS/MS analysis

759

760 **Mass-spectrometry analysis with the Thermofisher scientific Exploris-240**

761 The LC-MS/MS experiments were performed using an Ultimate 3000 RSLC nano system
762 (Thermo Fisher Scientific Inc, Waltham, MA, USA) interfaced online with a nano easy ion
763 source and an Exploris 240 Orbitrap mass spectrometer (Thermo Fisher Scientific Inc,
764 Waltham, MA, USA). The samples were analyzed in Data Dependent Acquisition (DDA). 6.5
765 µl of peptides were first loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5 µm
766 particle size, 100 Å pore size, 300 µm i.d. x 5 mm length) from the Ultimate 3000 autosampler
767 with 0.05% TFA in water at a flow rate of 10 µL/min. The peptides were separated by reverse-
768 phase column (Thermo Scientific PepMap C18, 2 µm particule size, 100 Å pore size, 75 µm i.d.
769 50 cm length)/ at a flow rate of 300 nl/min. After 3 min loading period, the column valve was
770 switched to allow elution of peptides from the pre-column onto the analytical column. Loading
771 buffer (solvent A) was 0.1% FA in water and elution buffer (solvent B) was 0.1% FA in 80%
772 acetonitrile. The linear gradient employed was 2-25% of solvent B in 103 min, then 25-40% of
773 solvent B from 103 to 123 min, finally 40-90% of solvent B from 123 to 125 min. The total run
774 time was 150 min including a high organic wash step and re-equilibration step. Peptides were
775 transferred to the gaseous phase with positive ion electrospray ionization at 1.9 kV. In DDA the
776 top 15 precursors were acquired between 350 and 1200 m/z with a 2 Th (Thomson) selection
777 window, dynamic exclusion of 40 s, normalized collision energy (NCE) of 30 and resolutions
778 of 120 000 for MS and 15 000 for MS2. Spectrum were recorded with Xcalibur software
779 (4.7.69.37) (Thermo Fisher Scientific).

780

781 **Identification and quantification**

782 The .raw files were analysed with MaxQuant version 2.0.3 using default settings. The minimal
783 peptide length was set to 6. The criteria “Trypsin/P” (which means C-terminus peptides of
784 “K/R” unless followed by “P”: “K/R” followed by “P” cannot be a cleavage site) was chosen
785 as the digestion enzyme. Carbamidomethylation of cysteine was selected as a fixed
786 modification and as variable modifications: N-terminal acetylation of protein; oxidation of
787 methionine; N-terminal-pyroglutamylation of glutamine and glutamate. Up to two missed
788 cleavages were allowed. The mass tolerance for the precursor was 20 and 4.5 ppm for the first
789 and the main searches, respectively, and for the fragment ions it was 20 ppm.

790 The files were searched against *Toxoplasma gondii* (March 2020 -https:
791 //www.uniprot.org/proteomes/UP000005641-8450 entries) and the Maxquant contaminant
792 database. Identified proteins were filtered according to the following criteria: at least two
793 different trypsin peptides with at least one unique peptide, an E value below 0.01 and a protein
794 E value smaller than 0.01 were required. Using the above criteria, the rate of false peptide
795 sequence assignment and false protein identification were lower than 1%. Proteins were
796 quantified by the label-free method with MaxQuant software using unique and razor peptide
intensities<sup>72</sup>. Statistical analyses were carried out using RStudio package software (t-test with p < 0.05 or 0.01 with or without Benjamini correction) to identify the significant differences in the protein abundance. Proteins were retained if they were quantified in at least two of the three replicates in at least one experiment.

Then the protein intensity ratio was calculated (protein intensity in test condition / protein intensity in control) to determine if has up-regulated or down-regulated. In some cases, certain proteins may be absent in one of the experimental conditions. To confirm the absence of a protein, we required that it be undetected in all three replicates of the given condition, while being consistently detected in all three replicates of the other condition. In such cases, protein presence or absence was determined based on the detection of unique peptides only.

## Transmission electron microscopy

HFF cells grown on Nunc Lab-tek chambers with Permanox bottom were infected with freshly egressed *T. gondii* tachyzoites following 24h of infection in presence or absence of IAA. The culture medium was replaced by the same volume of fixative (2.5% glutaraldehyde, in 0,1M sodium cacodylate pH7.4) and fixed for 2 hours at RT, then stored at 4°C in the fixative until further processing. The fixed cells were then washed twice in cacodylate buffer 0,1M to remove the glutaraldehyde. For scanning electron microscopy (SEM), embedding in Epon was performed using a modified version of the protocol of Hua et al.<sup>73</sup> All the following steps were performed using a Pelco Biowave Pro+ tissue processor (Ted Pella). The details of the program are indicated in Supplementary Table 5. Briefly, the cells were first fixed with a double osmium staining procedure as follows : **1)** 1% OsO<sub>4</sub>, 5 mM CaCl<sub>2</sub>, in cacodylate buffer 0,1 M pH 7.4; **2)** Cacodylate buffer washes; **3)** *In situ* reduction with potassium ferricyanide 1.5 %, 5mM CaCl<sub>2</sub> in cacodylate buffer; **4)** distilled water washes; **5)** 1% thiocarbohydrazide (TCH) in water; **6)** water washes **7)** 1% OsO<sub>4</sub> in water; **8)** distilled water washes. The samples were then
incubated in 2% uranyl acetate at 4°C overnight. The next day, the samples in uranyl acetate were heated 10 min at 37°C and processed with the Biowave (without washing). After processing, the samples were washed with water, incubated in lead aspartate, washed with water, and then dehydrated in growing concentrations of acetonitrile. Finally, they were progressively impregnated in EPON Hard⁺ resin directly into the Labtek. The resin was then polymerized at 60°C in an oven for 48 hours. TCH and lead aspartate were prepared extemporaneously as described in Deerinck et al.⁷⁴. For transmission electron microscopy (TEM), samples were simply stained with osmium (as above, step 1), incubated overnight in 2% uranyl acetate, and the dehydrated and epon embedded as for SEM.

Thin serial sections of 70 nm were prepared with a Leica UCT ultramicrotome using a 35° Jumbo ultra Diatome diamond knife. For SEM, sections were collected on silicon wafers that has been hydrophilized by glow discharge using a Safematic CCU-010 carbon coater equipped with an etching unit (30 seconds of plasma treatment at 8.10⁻¹ mbar, 15mA, in air). For TEM, sections were collected on 100 mesh copper grids coated with collodion and post-stained with uranyl acetate and lead citrate.

SEM images were acquired at the MRI-EM4Bio facility using a Zeiss Gemini 360 scanning electron microscope using the Volutome-BSD detector. The wafers were mounted on sample stubs using double-sided adhesive copper tape. Automated acquisitions were performed using Atlas 5 software for array tomography using the following parameters: voltage: 1.5 kV; aperture 30 µm; working distance 4 mm, dwell time 3.2µsec. Autofocus was applied once per tile and autostigmatism once per mosaic. Serial section series were aligned with Atlas and manually segmented using DragonFly 3D world software.

TEM images were acquired at the MEA facility on Jeol 1010 TEM equipped with a Quemsa Emsis camera at 80kv.

846

847 **Preparation of *Toxoplasma gondii* tachyzoites for cryo-electron tomography**
848 HFF cells were maintained in T25 flasks in Dulbecco's Modified Eagle Medium (DMEM;
849 Gibco) supplemented with 5% fetal calf serum (FCS) and 2 mM glutamine. Two confluent
850 flasks were infected with RCC1-2-mAID-HA *T. gondii* parasites strain. At 38 hours post-
851 infection, when parasites were fully intracellular, cultures were washed five times to remove
852 extracellular parasites and subsequently treated with or without indole-3-acetic acid (IAA).
853 After 10 hours of the treatment, freshly egressed tachyzoites were collected and resuspended in
854 phosphate-buffered saline (PBS) containing 10 nm gold fiducials. Approximately 4 × 10⁶
855 parasites in a total volume of 4 mL were applied to EM grids and plunge-frozen into a liquid
856 ethane/propane mixture using an EM GP2 automated plunger (Leica Microsystems, Wetzlar,
857 Germany) following 4-6 sec of front blotting.
858

858

859 **Cryo-electron tomography data collection and processing**
860 Cryo-electron tomography was performed on a Thermo Fisher Scientific Titan Krios G3i 300
861 keV field-emission cryo-transmission electron microscope equipped with an energy filter.
862 Dose-fractionated images were acquired using SerialEM software on a K3 direct electron
863 detector (Gatan) operated in electron-counting mode. Movie frames were motion-corrected
864 using the Alignframes function in IMOD. Tilt series were collected using a dose-symmetric
865 acquisition scheme over an angular range of -60° to +60° with 2° increments at a nominal
866 magnification of 33,000×, corresponding to a calibrated pixel size of 2.67 Å, and at a defocus
867 of approximately -5 µm. The cumulative electron dose per tilt series was approximately 120 e-
868 Å⁻². Tilt series alignment was performed through fiducial-based tracking in Etomo (IMOD
869 version 4.11.25) using the 10 nm colloidal gold bead as fiducial markers. The resulting
870 alignment parameters were subsequently imported into Warp (Version 2.0.0) for CTF
871 correction and reconstruction. Reconstructed tomograms were binned by a factor of 3.7,
872 yielding a final voxel size of 10 Å. Tomogram quality was further improved through denoising
873 with IsoNet2 (v2.0.0-beta). The resulting denoised tomograms were used for segmentation in
874 DragonFly (Version 2025.1).

875

876 **Conoid extrusion assay**

877 Tachyzoites pre-treated for 24h ± IAA were lysed by 3 passages through a 25-gauge needle,
878 then then added to poly-D-Lysine-coated coverslips pre-heated at 37°C. The plate was
879 centrifuged at 400 x g for 1 min to attach the parasites to the coverslips. Attached parasites were
880 incubated at 37°C for 8 min in pre-heated HEPES solution (274 mM NaCl, 10 mM KCl, 2 mM
881 Na₂HPO₄, 11 mM glucose, and 42 mM HEPES, pH 7.05) supplemented with 5 mM CaCl₂ and
882 5 µM A23187 calcium ionophore to induce the extrusion of conoid. As control, parasites
883 incubated in HEPES solution without A23187 were used. Standard U-ExM protocol was then
884 followed using anti-HA and anti-α/β tubulin primary antibodies.

885

886 **Ultrastructure expansion microscopy (U-ExM) of *Toxoplasma* tachyzoites**

887 Intracellular vacuoles in HFFs, or syringe-lysed tachyzoites seeded onto poly-D-lysine (Gibco)
888 coated coverslips, were transferred in a protein cross-linking solution (2% acrylamide/0.7%
889 formaldehyde in PBS) for 3 h at 37°C. Cells were then embedded in a gelation solution (19%
890 sodium acrylate/10% acrylamide/0.1% N,N'-methylenbisacrylamide/10% TEMED/10% APS
891 in PBS) on ice, prior to gelation for 1 h at 37°C. Gels were then incubated in denaturation buffer
892 (200 mM SDS, 200 mM NaCl, 50 mM Tris pH 9) for 15 min under agitation to facilitate their
893 detachment from coverslips. Gels were placed in Eppendorf tubes filled with denaturation
894 buffer and incubated at 95 °C for 90 min. Gels were then expanded overnight in ddH₂O at room
895 temperature, then shrank by two 15-min washes in PBS prior to antibody incubation. The gel
896 slices were incubated with primary antibodies diluted in 2% BSA/PBS at 37°C for 3 h. Gels
897 were then washed three times for 10 min in PBS containing 0.1% Tween 20, followed by
incubation with secondary antibodies at 37°C for 3 h. Gels were washed three times for 10 min in PBS containing 0.1% Tween 20 before the final expansion step, performed by incubating the gels in ddH₂O overnight. Gel slices were then placed onto poly-D-lysine-coated 24-mm coverslips and imaged with a Zeiss LSM880 confocal microscope equipped with Airyscan detector, with a 63x1.4 NA oil objective, using Zen Black software (Zeiss, Intelligent Imaging Innovations). Z-stacks were denoised, adjusted in brightness and contrast, colored, and processed to obtain maximum intensity projections using the Fiji software. Three-dimensional reconstructions of intracellular and extracellular parasites were generated using Imaris (version 9.9, Bitplane/Oxford Instruments). After confocal image stacks import, voxel size and channel parameters were set according to the acquisition metadata. Structures of interest (e.g., tubulin-based cytoskeleton, basal complex and RCC1-2 protein) were segmented using the Surface module based on their respective marker channel. For the subpellicular microtubules, the threshold level was determined from control images to include continuous SPMTs while excluding diffuse background. The resulting surfaces were visually inspected in 3D view and, where necessary, edited manually to remove spots outside the parasite.

913

914

915 Acknowledgements

We thank Sebastian Lourido for the pU6-Universal plasmid, and Dominique Soldati-Favre for providing the anti-ARM (ARO) antibodies and pLinker-2xTy-DHFR plasmid. We thank Lucien Daunas and Salomé Bellec from the MRI-EM4BIo facility and Franck Godiard from the MEA facility for their help in conducting experiments in ultrastructural electron microscopy. We are also grateful to Elodie Jublanc, Vicky Diakou and the imaging facility MRI at the University of Montpellier, member of the national infrastructure France-BioImaging (https://ror.org/01y7vt929) supported by the French National Research Agency (ANR-24-
923 INBS-0005 FBI BIOGEN), Christophe Duperray and Felicia Leccia of the MRI-Cytometry at
924 the Institute for Regenerative Medicine and Biotherapy for their assistance and technical
925 support. Mass spectrometry experiments were carried out using the facilities of the Montpellier
926 Proteomics Platform (PPM, MSPP site BioCampus Montpellier), a member of the national
927 Proteomics French Infrastructure (ProFI UAR 2048) supported by the French National
928 Research Agency (ANR-24-INBS-0015, Investments for the future F2030). We thank Shrawan
929 Kumar Mageswaran for his technical assistance with cryo-electron tomography, and the Singh
930 Center for Nanotechnology and the Beckman Center for Cryogenic Electron Microscopy at the
931 University of Pennsylvania for hosting and supporting the use of the Titan Krios cryo-electron
932 microscope. Dr Maryse Lebrun is an INSERM researcher. Dr Mathieu Gissot is a CNRS
933 researcher. This work was supported by the Laboratoire d'Excellence (LabEx) (ParaFrap ANR-
934 11-LABX-0024), an European Research Council (ERC advanced grant number 833309
935 KissAndSpitRhoptry) to M.L.; by the OPENSESAME for Apicomplexa INSERM IPR project
936 to Y-W.C. and M.L.; by the Pasteur Network grant (ACIP 76-2017 and PTR-474-21) to M.G;
937 by a National Institute of Health R35 Award (R35GM156396) and a Burroughs Wellcome Fund
938 Investigators in the Pathogenesis of Infectious Disease Program Award (1022785) to Y.-W.C.
939 M.B. is supported by the European Research Council (ERC) under the European Union's
940 Horizon 2020 research and innovation program under Grant agreement no. 833309 to M.L.

941

942 Author contributions

943 Conceptualization: M.B., M.L., M.G. Investigation: M.B., A.B., M.P. and T. M. generated the
944 tagged and iKD lines and analyzed transgenic lines; M.B. performed phenotypic analysis of the
945 mutants with the help of M.P., A.B., M.M., J.D, and T.M.; M.B. generated and analyzed the
946 TurboID line; M.B. and A.B. performed the TurboID experiments and V.R. performed the mass
947 spectrometry experiments and analysis; M.B. performed all ultrastructure expansion
948 microscopy; J.F.D., A.G. and L.B.-S. performed EM analysis; Q.N. and S.P. performed cryo-

949 ET. Visualization: M.B, M.P, Q.N., S.P., Y.-W.C. M.G, M.L. Writing-Review & Editing: M.B.,

950 M.G., M.L., Y.-W.C., with inputs from all authors. Supervision: Y.-W.C., M.G., M.L. Funding:

951 Y.-W.C., M.G., M.L.

952

953 **Declaration of interests**

954 The authors declare no competing interests

955

956 **Data availability**

957 The mass spectrometry proteomics data have been deposited to the ProteomeXchange

958 Consortium via the PRIDE 75 partner repository with the dataset identifier PXD074368.

959

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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
FIGURE LEGENDS

**Figure 1.** TgRCC1-2 is recruited at the apical end of daughter cells throughout endodyogeny.

**a,** Schematic representation of *T. gondii* cell division by endodyogeny. The initiation step begins with centrosome duplication (red dot), followed by nucleation, during which DCS starts to assemble concurrently with the formation of nascent preconoidal ring (PCR; blue ring), conoid (green) and apical polar ring (APR; red ring). During the elongation step, the inner membrane complex (IMC, light blue) continues to elongate, and the nucleus (blue) adopts a characteristic U-shape prior to fission. In the final constriction step, the mother plasma membrane envelops the emerging daughter cells, while the residual maternal components are disassembled and degraded. **b,** Assessment of protein depletion in RCC1-2-mAID-HA₃ cell lines by western blot following IAA treatment for 0, 4, or 6 hours. TgROP5 was used as loading control. **c,** Representative images of plaque assays for RCC1-2mAID-HA₃ tagged line in the absence or presence of IAA. **d,** Schematic representation of the TgRCC1-2 protein, highlighting RCC1-repeats at the N-terminus (orange) and C-terminal coiled coil domains (green). **e,** Localization of RCC1-2 and ISP1 by confocal microscopy. Endogenously double tagged RCC1-2-mAID-HA₃ and ISP1-Ty2 parasites were stained with anti-HA (green) and anti-Ty (magenta) antibodies to visualize RCC1-2 and the apical cap marker ISP1, respectively. DNA is labeled with Hoechst. White arrowheads point at RCC1-2 signal. Dotted outlines indicate individual parasites, with mothers shown in white and daughters in light blue. Shown are maximum-intensity projections of z-stack confocal images. Scale bars are 2µM. **f,** Confocal immunofluorescence images of RCC1-2-mAID-HA₃ parasites, labeled with anti-HA (green) and anti-IMC1 (magenta) antibodies to visualize RCC1-2 and the IMC, respectively. DNA is labeled with Hoechst. White arrowheads point at RCC1-2 signal. Shown are maximum-intensity projections of z-stack confocal images. Scale bars are 2µM. **g,** U-ExM images of tachyzoites expressing RCC1-2 throughout endodyogeny. Parasites were stained with anti-HA
(green) and anti-α/β tubulin (magenta) antibodies to label RCC1-2 and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight daughter cell buds. White-contoured arrowheads indicate mother apical complex (M: mother). White arrowheads point developing daughter cells structures (D: daughter) (Ce: centrioles; S: spindle; ICMT: intraconoidal microtubules; Co: conoid). Scale bars are 5μM. Yellow arrowheads mark the five rafts of nascents SPMTs. h, U-ExM images of RCC1-2 in dividing parasites. Parasites were stained with anti-HA (red), anti-Centrin1 (green) and anti-α/β-tubulin (white) antibodies to label RCC1-2, centrioles and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight centrosomes of mother cell. Black arrowheads indicate mother apical complex (M: mother). White-contoured arrowheads point developing daughter cells structures (D: daughter) (Ce: centrioles; S: spindle). Scale bars: 5μM.
<figure><img src="image_3.png" /><figcaption>b</figcaption></figure>

RCC1-2-mAID-HA<sub>3</sub> + CPH1-Ty

<figure><img src="image_4.png" /></figure>

Merge

<figure><img src="image_5.png" /></figure>

Inset

<figure><img src="image_6.png" /></figure>

Merge

RCC1-2-mAID-HA<sub>3</sub> + CPH1-Ty

<figure><img src="image_7.png" /></figure>

Merge

<figure><img src="image_8.png" /></figure>

Inset

Side view

<caption>c</caption>

RCC1-2-mAID-HA<sub>3</sub>

<figure><img src="image_9.png" /></figure>

HA

Tub

HA

Tub

Extruded conoid

<figure><img src="image_10.png" /></figure>

HA

Tub

HA

Tub

<caption>d</caption>

RCC1-2-mAID-HA<sub>3</sub> + KinA-Ty

<figure><img src="image_11.png" /></figure>

Merge

Inset

<figure><img src="image_12.png" /></figure>

Merge

RCC1-2-mAID-HA<sub>3</sub> + KinA-Ty

<figure><img src="image_13.png" /></figure>

Merge

<figure><img src="image_14.png" /></figure>

Inset

Side view

<caption>e</caption>

RCC1-2-mAID-HA<sub>3</sub> + APR7-Ty

<figure><img src="image_15.png" /></figure>

Merge

Inset

<figure><img src="image_16.png" /></figure>

Merge

RCC1-2-mAID-HA<sub>3</sub> + APR7-Ty

<figure><img src="image_17.png" /></figure>

Merge

<figure><img src="image_18.png" /></figure>

Inset
**Figure 2. RCC1-2 localizes below the bottom layer APR.** *a*, Schematic representation of *T. gondii* apical complex in retracted conoid state (adapted from Ren *et al.*¹⁶). *b*, U-ExM images of intracellular tachyzoites showing the localization of RCC1-2 (red), the Ty-tagged conoid protein, CPH1 (green) and α/β tubulin (white). Images represent maximum-intensity projections of z-stack confocal sections, with both top and side views are left and right panels, respectively. Insets highlight the apical complex of a single developing daughter cell. Scale bars are 5µM. *c*, U-ExM images of RCC1-2 localization and tubulin (magenta) in syringed-lysed tachyzoites with extruded conoid state. Images represent z-stack confocal sections. Scale bars are 1µM. RCC1-2 localized beneath the extruded conoid, extending into SPMTs interspaces. *d-e*, U-ExM images of intracellular tachyzoites showing the localization of RCC1-2, tubulin and Ty-tagged bottom layer APR proteins, KinA (*d*) and APR7 (*e*). Images represent maximum-intensity projections of z-stack confocal sections, with both top and side views shown (left and right panels, respectively). Insets highlight the apical complex of a single developing daughter cell. Scale bars are 5µM. RCC1-2 localized below the last known layer of the APR. *b*, *d*, *e*) DNA is labeled by Hoechst (blue).
<figure><img src="image_19.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>IMC</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Gliosomes</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>d</figcaption></figure>
**Figure 3.** Loss of RCC1-2 causes structural defects of the pellicle. **a**, Confocal immunofluorescence images of RCC1-2-mAID-HA₃ parasites costained with IMC1, GAP45 and SAG1 antibodies, or RCC1-2-mAID-HA₃ parasites expressing ISP1-Ty2, left untreated or treated with IAA for 24h. DNA is labeled with Hoechst. Scale bars are 2µM. **b-c**, Quantification of phenotypical defects displayed in panel (a). **b**, Rosette and disorganized vacuoles were quantified from approximately 100 vacuoles and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA test. **c**, Non-dividing, synchronous and asynchronous dividing vacuoles were quantified from approximately 300 vacuoles and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA test. Vacuoles containing parasites at multiple division stages were scored as asynchronous. **d**, Selected electron micrographs from 70 nm serial sections of dividing RCC1-2-depleted tachyzoites. The section number is indicated at the top left corner. 1st row: the IMC of the daughter cells showed multiple interruptions (yellow asterisks), partial overlapping of IMC sheets and isolated IMC fragments (yellow arrowhead). The second row shows the same image after segmentation of the cellular compartments. Daughter IMC (pink), nucleus (blue), conoid (green). Scale bars are 1 µm.
<caption>a</caption>

Intracellular parasites
RCC1-2-mAID-HA₃

<figure><img src="image_24.png" /><figcaption>b</figcaption></figure>

Extracellular parasites
RCC1-2-mAID-HA₃

<figure><img src="image_25.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>d</figcaption></figure>

DOC extraction
RCC1-2-mAID-HA₃

<figure><img src="image_27.png" /><figcaption>e</figcaption></figure>

Intracellular parasites
RCC1-2-mAID-HA₃

<figure><img src="image_28.png" /><figcaption>f</figcaption></figure>

RCC1-2-mAID-HA₃
+ CPH1-Ty

<figure><img src="image_29.png" /><figcaption>g</figcaption></figure>

RCC1-2-mAID-HA₃
+ KinA-Ty

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>h</figcaption></figure>

RCC1-2-mAID-HA₃
+ APR7-Ty

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

-IAA
Ty
Tub

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

-IAA
+IAA

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

-IAA
+IAA

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

Ctrl

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

RCC1-2-mAID (+IAA)

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

IMC

Top APR layer

APR layers

Top
Middle
Bottom

SPMTs
**Figure 4. RCC1-2 is required for SPMTs anchoring to the APR. a-c) SPMTs organization in RCC1-2-depleted tachyzoites.** U-ExM images of intracellular (a) or syringed-lysed tachyzoites (b) left untreated or treated with IAA for 24h. Tachyzoites were stained with anti-HA (green), anti-α/β tubulin antibodies (magenta) to label RCC1-2 and microtubules respectively. DNA is labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Scale bars are 5μM. **c,** Quantification of SPMTs defects displayed in panel (b). SPMTs number was quantified from approximately 20 parasites and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by one-way ANOVA test. **d,** Images of freshly egressed parasites subjected to DOC extraction. Parasites were labeled with an anti-HA (magenta) and anti-acetylated tubulin (green) antibodies to label RCC1-2 and microtubules, respectively. Scale bars are 2μM. **e,** U-ExM images of dividing tachyzoites, either left untreated (left panel) or IAA-treated (right panel) for 24h. Tachyzoites were stained with anti-α/β tubulin antibodies (magenta) to label microtubules and Hoechst (blue) for DNA. Images represent maximum-intensity projections of z-stack confocal sections. White-contoured arrowheads indicate mother conoid (mCo). White arrowheads point daughter cells structures (dCo: daughter conoid; dSPMTs: daughter subpellicular microtubules). Scale bars are 2μM. **f-h,** Representative images of apical markers in daughter cells by U-ExM. U-ExM images of RCC1-2-mAID-HA₃ + CPH1-Ty2 (f), + KinA-Ty2 (g) and + APR7-Ty2 (h) parasites, either left untreated or treated with IAA for 24h. Intracellular tachyzoites were stained with anti-Ty (green) and anti-α/β tubulin (magenta) antibodies to label Ty-tagged proteins and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. White arrows indicate apical complex of daughter cells. Disruption of the targeted marker was quantified from approximately 60 parasites and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA test. Scale bars are 1μM. **i-n,**
Cryo-ET tomograms of control and RCC1-2-depleted parasites. (i-j) Tomogram slices of WT (i) and RCC1-2-depleted parasites (j) both show the inner membrane complex (IMC, green) anchored to the top layer of the apical polar ring (APR, blue), as detailed in the enlarged insets (i', j'). (k-l) Tomogram slices of the apical end show that the top (blue arrow), middle (white arrow), and bottom (orange arrow) layers of the APR exhibit band-like appearance in both WT (k, k') and RCC1-2-depleted parasites (l, l'). (m-n) Tomogram slices showing SPMT attachment sites on APR in WT (m, m') and RCC1-2-depleted (n, n') parasites. SPMTs (magenta); SPMT detached from the APR (magenta arrow); interspersed pillars densities franked by attached SPMTs (yellow arrows); interspersed pillars densities next to the detached SPMTs from the APR (red arrows). Scale bars: 100 nm
<figure><img src="image_46.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_51.png" /></figure>
**Figure 5. Proximity labelling of RCC1-2 identifies novel daughter-specific proteins. a,**
Confocal immunofluorescence images of RCC1-2-HA3-TurboID parasites after 0 or 2 hours of biotin treatment. White arrowheads point at RCC1-2 signal. Parasites were labeled with anti-HA (green) and streptavidin-based fluorophore (magenta) to label RCC1-2 and biotinylated sites, respectively. Scale bars are 2µM. **b,** Western blot analysis of immunoprecipitated proteins with anti-streptavidin beads from RCC1-2-HA3-TurboID parasites untreated or treated with biotin for 2 hours. Samples were immunoblotted with anti-HA (upper panel), anti-streptavidin-HRP (middle panel). TgROP5 was used as IP negative control (lower panel). **c,** Schematic representation of the size of the three RCC1-2 proximity candidates and their INTERPRO-annotated functional domains⁵¹. Coiled-coil domain (green). TOG: Tumor Overexpression Gene,. TTL: Tubulin Tyrosine Ligase domain. **d,** Confocal immunofluorescence images of the three candidate proteins fused with a mAID-HA3 cassette (-mAID for short) in non-dividing (upper panel) and dividing parasites (lower panel). Parasites were stained with anti-HA (green) and anti-IMC1 (magenta) antibodies to visualize HA-tagged proteins and the IMC, respectively. White arrowheads point at apical signal. Scale bars are 2µM. **e-f,** Assessment of fitness and protein depletion of the three retained candidates. Immunoblot analysis (**e**, right panel) using an anti-HA antibody was performed on lysates from intracellular parasites (mAID-HA tagged lines) that were either untreated or treated with IAA for 24h. TgROP5 was used as loading control. Images of plaque assay (**e**, left panel) and quantification of plaques areas (**f**) for control (Tir1) and mAID-HA3 tagged lines in the absence or presence of IAA. Values are reported as mean ± SD (n = 3 biological replicates, each with three technical replicates). Statistical significance was determined by unpaired two-tailed Student's t-test. RCC1-proximity candidates were rapidly degraded upon addition of IAA and depletion resulted in no visible plaque formation after 7 days only for TGGT1_212780.
212780-mAID-HA<sub>3</sub>

212780-mAID-HA<sub>3</sub>

<figure><img src="image_52.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>c</figcaption></figure>

212780-mAID-HA<sub>3</sub> + PCR1-Ty

212780-mAID-HA<sub>3</sub> + CPH1-Myc

212780-mAID-HA<sub>3</sub> + APR3-Ty

212780-mAID-HA<sub>3</sub> + APR2-Ty

<figure><img src="image_54.png" /><figcaption>Conoid</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>Upper layer APR</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>Middle layer APR</figcaption></figure>

<figure><img src="image_57.png" /></figure>

212780-mAID-HA<sub>3</sub> + KinA-Ty

212780-mAID-HA<sub>3</sub> + APR7-Ty

212780-mAID-HA<sub>3</sub> + RCC1-2-Ty

<figure><img src="image_58.png" /><figcaption>Bottom layer APR + IMC</figcaption></figure>

<figure><img src="image_59.png" /><figcaption>RCC1-2</figcaption></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /><figcaption>d</figcaption></figure>

212780-mAID-HA<sub>3</sub> + PCR1-Ty

<figure><img src="image_62.png" /></figure>

212780-mAID-HA<sub>3</sub> + KinA-Ty

<figure><img src="image_63.png" /><figcaption>Bottom layer APR</figcaption></figure>

212780-mAID-HA<sub>3</sub> + APR3-Ty

212780-mAID-HA<sub>3</sub> + APR7-Ty

<figure><img src="image_64.png" /><figcaption>Bottom layer APR +IMC</figcaption></figure>

<figure><img src="image_65.png" /></figure>

212780-mAID-HA<sub>3</sub> + APR2-Ty

212780-mAID-HA<sub>3</sub> + RCC1-2-Ty

<figure><img src="image_66.png" /><figcaption>RCC1-2</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>Middle layer APR</figcaption></figure>
**Figure 6.** TGGT1_212780 is one of the earliest proteins expressed at the bottom layer APR during endodyogeny. **a,b** U-ExM images of dividing tachyzoites expressing TGGT1_212780-mAID-HA₃ during endodyogeny. Parasites were stained with anti-HA (green) and anti-α/β tubulin (magenta) antibodies to label TGGT1_212780 and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight developing daughter cells. White-contoured arrowheads indicate mother apical complex (M: mother). White arrowheads point developing daughter cells structures (D: daughter) (Ce: centrioles; S: spindle; Co: conoid). Yellow arrowheads mark the five rafts of nascents SPMTs. Scale bars are 5µM. **c,** U-ExM images of TGGT1_212780-mAID-HA₃ in combination with multiple Ty/Myc-tagged apical complex proteins, shown as side view of a single daughter cell. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty or anti-Myc (green) and anti-α/β tubulin (white) antibodies to label TGGT1_212780, apical markers and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. Scale bars are 1µM. Bottom right: schematic showing apical complex markers in daughter cell: the PCR marker PCR1, the conoid protein CPH1 and the multilayered APR (from top to bottom: APR3, APR2, KinA, APR7 and RCC1-2). TGGT1_212780 is a daughter APR protein associated with the bottom layer of the APR. **d,** U-ExM images showing TGGT1_212780-mAID-HA₃ in combination with multiple Ty-tagged apical complex proteins at early stage of endodyogeny. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty or anti-Myc (green) and anti-α/β tubulin (white) antibodies to label TGGT1_212780, apical markers and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight developing daughter cells. Black arrows indicate mother apical complex (M: mother). White-contoured arrowheads point developing daughter cells structures
(D: daughter) (Ce: centrioles; S: spindle; Co: conoid). Yellow arrowheads mark the five rafts of nascents SPMTs. Scale bars are 5µM.
<figure><img src="image_68.png" /><figcaption>a. APR8-mAID-HA<sub>3</sub></figcaption></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /></figure>
**Figure 7.** APR8 depletion prevents correct assembly of IMC and SPTMs. **A**, Western blot showing protein depletion in APR8-mAID-HA₃ parasites following IAA treatment for 0, 4, or 6 hours. TgROP5 was used as loading control. APR82 was already undetectable within 4 h of IAA. **b-c**, SPMTs organization in APR8-depleted tachyzoites. U-ExM images of dividing tachyzoites, either left untreated (left panel) or IAA-treated (right panel) for 6h. Tachyzoites were stained with anti HA (green), anti-α/β tubulin antibodies (magenta) to label APR8 and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. White-contoured arrowheads indicate mother conoid (mCo). White arrowheads point daughter cells structures (dCo: daughter conoid; dSPMTs: daughter subpellicular microtubules; S: spindle). Scale bars are 2μM. **c**, Quantification of SPMTs defects in daughter cells displayed in panel (b). SPMTs disruption was quantified from approximately 80 daughter cells and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA test. **c**, Confocal immunofluorescence images of the IMC in APR8-mAID-HA₃ parasites, either left untreated or treated with IAA for 24h. Intracellular tachyzoites were stained with anti-IMC1 (green) and Hoechst (blue) to label the IMC and nuclear DNA, respectively. Scale bars are 2μM. Disruption of the IMC was quantified from approximately 100 vacuoles and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA test. **d-e**, Representative transmission electron microscopy (TEM) images of APR8-depleted tachyzoites. Panel **d** shows the whole mother cell upon endodyogeny while panel **E** focuses on daughter cells. White arrows highlight discontinuous flotting IMC of daughter cells (dIMC: daughter IMC; mIMC: mother daughter cell; N: nucleus). Scale bars: 2um (d), 0,5um (e).
<caption>Figure 8</caption>

a

<figure><img src="image_75.png" /></figure>

b

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /><figcaption>c APR8-mAID-HA<sub>3</sub> + CPH1-<span style="color:red;">Myc</span> + PCR1-<span style="color:red;">Ty</span> +IAA</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>d APR8-mAID-HA<sub>3</sub> + CPH1-<span style="color:red;">Myc</span> + APR3-<span style="color:red;">Ty</span> +IAA</figcaption></figure>

<figure><img src="image_82.png" /></figure>

e

<figure><img src="image_83.png" /><figcaption>APR8-mAID-HA<sub>3</sub> + CPH1-<span style="color:red;">Myc</span> + APR2-<span style="color:red;">Ty</span> +IAA</figcaption></figure>

f

<figure><img src="image_84.png" /><figcaption>RCC1-2-mAID-HA<sub>3</sub> + APR8-Ty</figcaption></figure>

g

<figure><img src="image_85.png" /><figcaption>-RCC1-2</figcaption></figure>

<figure><img src="image_86.png" /><figcaption>-APR8</figcaption></figure>

<figure><img src="image_87.png" /></figure>
**Figure 8.** APR8 acts as a scaffold for APR organization and subsequent proper apical complex development. **a-b,** Apical markers expression in APR8-mAID-HA₃ strain. **a,** Left: Schematic showing apical complex markers quantified in daughter cell: the PCR marker PCR1, the conoid protein CPH1 and the multilayered APR (from top to bottom: APR3, APR2, KinA, APR7 and RCC1-2). Right: Quantification of apical markers in APR8-mAID-HA₃ in combination with Ty-/Myc-tagged apical markers, left untreated or IAA-treated for 6h. Presence of apical markers was quantified from approximately 50 daughter cells exhibiting elongated SPMTs and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by unpaired two-tailed Student's t-test. **b,** Representative U-ExM images of daughter apical complex in APR8-mAID-HA₃ strain with Ty-/Myc-tagged apical markers, either left untreated or IAA-treated for 6h. Intracellular tachyzoites were stained with anti-Ty or anti-Myc (green) and anti-α/β tubulin (magenta) antibodies to label apical markers and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections, with inset zoom highlighting a single daughter apical complex. White arrowheads point daughter conoid. Scale bars are 1µM. **c-e,** Representative U-ExM images of apical markers in triple epitope-tagged APR8-mAID-HA₃ strains following IAA depletion for 4h. Apical complex markers proximity was investigated in APR8-mAID-HA₃ depleted parasites expressing CPH1-Myc-depleted tachyzoites in combination with either the PCR marker PCR1 (c), the upper-layer APR protein APR3 (d), or the middle-layer APR component APR2 (e). Intracellular tachyzoites were immunostained with anti-Myc (green) to label the conoid protein CPH1, anti-Ty (red) for Ty-tagged proteins, and anti-α/β-tubulin (white) for microtubules. Images represent maximum-intensity projections of z-stack confocal sections. White-contoured arrowheads point the mother apical complex (M: mother). Insets highlight developing daughter cells with white arrowheads indicating their apical complex (D: daughter). Scale bars are 2µM. **f,** Representative U-ExM images of RCC1-2-mAID-HA3 depleted
parasites expressing APR8-Ty. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty (green) and anti-α/β tubulin (white) antibodies to label RCC1-2, APR8 and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. White-contoured arrowheads point the mother apical complex (M: mother). Insets highlight developing daughter cells (D: daughter). Scale bars are 5μM. Right: Quantification of APR8 signal at daughter apical complex upon RCC1-2 depletion. Presence of APR8 was quantified from approximately 30 daughter cells at early stage of endodyogeny and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by unpaired two-tailed Student's t-test g, Schematic model illustrates a dividing tachyzoite with a zoomed inset on daughter apical complex organization under wild-type, RCC1-2-depleted and APR8-depleted conditions. RCC1-2 is positioned just beneath the bottom layer of the APR, whereas APR8 is a resident protein of the bottom layer APR. Both RCC1-2 and APR8 function as critical scaffolding proteins that govern distinct stages of DCS budding during endodyogeny. In RCC1-2-depleted tachyzoites, detached SPMTs are evident, demonstrating that RCC1-2 is essential for proper anchoring and stabilization of SPMTs to the APR.
<figure><img src="image_88.png" /><figcaption>a</figcaption></figure>

<figure><img src="image_89.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_90.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_91.png" /></figure>

<figure><img src="image_92.png" /></figure>

<figure><img src="image_93.png" /></figure>

<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

<table><thead><tr><th></th><th>RCC1-2</th><th>RCC1-3</th><th>RCC1-4</th><th>RCC1-5</th><th>RCC1-6</th><th>RCC1-7</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>2865bp</td><td>3042bp</td><td>3035bp</td><td>3000bp</td><td>3233bp</td><td>2908bp</td></tr><tr><td>2: P1+P2</td><td>348bp</td><td>384bp</td><td>523bp</td><td>375bp</td><td>523bp</td><td>362bp</td></tr><tr><td>3: P3+P4</td><td>763bp</td><td>815bp</td><td>758bp</td><td>870bp</td><td>956bp</td><td>792bp</td></tr><tr><td>Ctrl P1+P4</td><td>291bp</td><td>484bp</td><td>340bp</td><td>395bp</td><td>520bp</td><td>192bp</td></tr></tbody></table>
**Supplementary Figure 1. Characterization of RCC1-repeats containing proteins in *T. gondii*.** **a**, Schematic representation of the RCC1-repeats containing proteins expressed during S/M phase and their INTERPRO-annotated functional domains⁵¹. RCC1: regulator of chromosome condensation 1-like domains (RLDs) (shown in orange). HECT: Homologous to the E6-AP Carboxyl Terminus (shown in blue), a catalytic domain found in E3 ubiquitin-protein ligases⁶². Coiled-coil domain⁶³ (shown in green). RanBP2: RanBP2 type zinc finger domain (shown in purple), a prevalent domain found in nucleoporins and splicing factors⁶⁴. MYOSIN: myosin motor domain (shown in pink), a conserved motor head domain that binds ATP and actin filaments to generate force and movement⁶⁵. **b**, Illustration of the cloning strategy employed to obtain the -mAID-HA₃ strain using CRISPR/Cas9 (top panel). Orange arrows depicted the designed primers used to validate the successful integration at the endogenous locus. **C**, Validation of successful integration of the mAID-HA₃ cassette into *TgRCC1-2* (*TGGT1_280770*), *TgRCC1-3* (*TGGT1_313570*), *TgRCC1-4* (*TGGT1_313860*), *TgRCC1-5* (*TGGT1_222430*), *TgRCC1-6* (*TGGT1_209130*), *TgRCC1-7* (*TGGT1_239700*) locus by PCR. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected PCR product sizes for each primer pair.
<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /></figure>

<figure><img src="image_101.png" /></figure>
**Supplementary Figure 2. Conditional depletion of RCC1-repeats candidates.** **a**, Confocal immunofluorescence images of the six RCC1-repeats containing proteins fused with a mAID-HA cassette (-mAID for short) in non-dividing (upper panel) and dividing parasites (lower panel). Parasites were stained with anti-HA (green) and anti-IMC1 (magenta) antibodies to visualize HA-tagged proteins and the IMC, respectively. Scale bars are 2µM. **b**, Same as **a**, in dividing parasites upon IAA depletion. Scales bars are 2µM. **c-d**, Assessment of fitness and protein depletion of the six RCC1-repeats containing proteins. **c**, Representative images of plaque assays for mAID-HA₃ tagged lines in the absence or presence of IAA and their corresponding immunoblot analysis, using an anti-HA antibody on lysates from intracellular parasites (mAID-HA tagged lines) that were either untreated or treated with IAA for 24h. TgAMA1 or TgROP5 served as a loading control. Protein molecular weight (in kDa) are shown on the left of each panel. **D**, Quantification of plaques areas for control (Tir1) and mAID-HA₃ tagged lines in the absence or presence of IAA. Values are reported as mean ± SD (n=3 biological replicates, each with three technical replicates). Statistical significance was determined by unpaired two-tailed Student's t-test. RCC1-repeats containing proteins were rapidly degraded upon addition of IAA and depletion resulted in no visible plaque formation after 7 days only for RCC1-2.
<figure><img src="image_102.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_103.png" /></figure>

<table><caption>c</caption><thead><tr><th></th><th>ISP1</th><th>CPH1</th><th>APR7</th><th>KinA</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>4914bp</td><td>4745bp</td><td>4724bp</td><td>4758bp</td></tr><tr><td>2: P1+P2</td><td>408bp</td><td>189bp</td><td>226bp</td><td>191bp</td></tr><tr><td>3: P3+P4</td><td>936bp</td><td>985bp</td><td>927bp</td><td>996bp</td></tr><tr><td>Ctrl P1+P4</td><td>501bp</td><td>336bp</td><td>244bp</td><td>438bp</td></tr></tbody></table>

<figure><img src="image_104.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_105.png" /></figure>

Lanes:

1: RCC1-2-mAID-HA<sub>3</sub> + ISP1-Ty2

2: RCC1-2-mAID-HA<sub>3</sub> + CPH1-Ty2

3: RCC1-2-mAID-HA<sub>3</sub> + APR7-Ty2

4: RCC1-2-mAID-HA<sub>3</sub> + KinA-Ty2
**Supplementary Figure 3. Generation of double-tagged strains in RCC1-mAID-HA₃ strain.** **a**, Illustration of the cloning strategy employed to obtain the double epitope-tagged RCC1-2-mAID-HA₃ strain using CRISPR/Cas9. Orange arrows depicted the designed primers used to validate the successful integration at the endogenous locus. **b**, Validation of successful integration of the TY2-DHFR cassette into *TgISP1*, *TgCPH1*, *TgAPR7* and *TgKinA* locus by PCR within the RCC1-2-mAID-HA₃ background. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected PCR product sizes for each primer pair. **c**, Validation of successful expression of ISP1-Ty2, CPH1-Ty2, APR7-Ty2 and KinA-Ty2 in RCC1-2-mAID-HA₃ background. Immunoblot analysis using an anti-HA and anti-Ty antibodies was performed on lysates from intracellular parasites. TgROP5 was used as loading control. Protein molecular weights (in kDa) are indicated on the left of each panel. **d**, U-ExM images showing RCC1-2-mAID-HA₃ in combination with either KinA-Ty2 or APR7-Ty2 at early stage of endodyogeny. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty (green) and anti-α/β tubulin (white) antibodies to label RCC1-2, apical markers and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight developing daughter cells. White-contoured arrowheads indicate mother apical complex (M: mother). White arrowheads point developing daughter cells structures (D: daughter) (Ce: centrioles; S: spindle; Co: conoid). Yellow arrowheads mark the five rafts of nascents SPMTs. Scale bars are 5µM.
<figure><img src="image_106.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_107.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_108.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_109.png" /></figure>
**Supplementary Figure 4. RCC1-2 is essential for the ultrastructure organization and not for apical secretory organelles positioning. a-b,** Confocal immunofluorescence images of RCC1-2-mAID-HA₃ parasites co-labeled for apical secretory organelles. Parasites were labeled with anti-HA (red) to label RCC1-2 and with either anti-AMA1 for micronemes (a) or anti-ARO for rhoptries (b) (green). Nuclear DNA was labeled with Hoechst (blue). Upper and lower panels show untreated and IAA-treated parasites for 24 hours, respectively, presented as maximum-intensity projections of z-stack confocal sections. Scale bars are 2µM. **c,** Representative electron micrographs of dividing RCC1-2-depleted tachyzoites. Black arrows highlight interrupted mother IMC (mIMC). Scale bars are 1µm. **d,** IMC Continuity. Tomogram slice demonstrates the preservation of the IMC (green) in WT parasites with intact plasma membrane (left panel). In contrast, RCC1-2 iKD (right panel) results in focal breakage and loss of structural continuity along the IMC (green) in parasites with intact PM, showing disintegration from the double membrane structure into a highly dense single membrane structure (inset). Scale bars: 100 nm.
<figure><img src="image_110.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_111.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_112.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_113.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_114.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_115.png" /></figure>
**Supplementary Figure 5. Ultrastructural defects of RCC1-2 depleted parasites impede parasite motility and invasion.**

**a,** Imaris 3D reconstruction of U-ExM images from syringed-lysed tachyzoites, either left untreated or IAA-treated for 24h. Tachyzoites were stained with anti-α/β tubulin (magenta) antibody and Hoechst (blue) to label microtubules and nuclear DNA, respectively. Images represent maximum-intensity projections of z-stack confocal sections with both side and top views (left and right panels, respectively). White arrows indicate mother apical complex. SPMT numbers annotated on the top-view tachyzoite images. Scale bars are 1µM. **b,** Representative images of apical markers in mother cells by U-ExM. U-ExM images of RCC1-2-mAID-HA₃ + CPH1-Ty2 (**f**), + KinA-Ty2 (**g**) and + APR7-Ty2 (**h**) parasites, either left untreated or treated with IAA for 24h. Intracellular tachyzoites were stained with anti-Ty (green) and anti-α/β tubulin (magenta) antibodies to label Ty-tagged proteins and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. White arrows indicate mother apical complex. Scale bars are 2µM. RCC1-2 depletion leads to loss of APR7 integrity. **c,** Gliding motility is impaired upon RCC1-2 depletion: Parasites were treated with A23187 to stimulate gliding motility visualized by gliding trails labeled with an SAG1 antibody. Scale bars are 5µM. **d,** RCC1-2 depletion is responsible of an invasion defect: Invasion was monitored for control (Tir1) and RCC1-2-depleted tachyzoites upon 24 h treatment with IAA. Values are reported as mean ± SD (n = 3 biological replicates, each with three technical replicates). Statistical significance was determined by unpaired two-tailed Student's t-test. **e,** Stimulated parasite egress is not affected in the absence of RCC1-2: Infected cells were treated with A23187 to stimulate parasite egress, measured as a number of egressed parasitophorous vacuoles over the total number. Egress was tested for control (Tir1) and RCC1-2-mAID-HA₃ cell lines in the absence of IAA, and upon 24h of IAA treatment. Values are reported as mean ± SD (n = 3 biological replicates, each with three technical replicates). Statistical significance was determined by unpaired two-tailed Student's t-test. At least 100
PVs were quantified in each replicate. P-values are non-significant for all datasets (two-tailed t-test). **f**, Representative images of egress assay treated as in **e**. Parasites were stained with anti-GAP45 (red) and anti-GRA3 (green) antibodies to label parasites plasma membrane and parasitophorous vacuoles, respectively. DNA is labeled with Hoechst (blue). Scale bars are 2µM.
a

RCC1-2-mAID-HA<sub>3</sub> -IAA

Early nucleation

Merge

HA
MORN1
Tub
Hoechst

Tub

Hoechst

Inset

Co

S

MORN1

Tub

Merge

Early nucleation

RCC1-2-mAID-HA<sub>3</sub> +IAA

Inset

Co

S

MORN1

Tub

Merge

Late nucleation

HA
MORN1
Tub
Hoechst

Tub

Hoechst

Tub

Hoechst

Tub

Hoechst

Tub

Inset

S

Co

MORN1

Tub

Merge

Late nucleation

HA
MORN1
Tub
Hoechst

Tub
Hoechst

Inset

Co

MORN1

Tub

Merge

Elongation

Merge

HA
MORN1
Tub
Hoechst

Tub

Hoechst

Inset

Co

MORN1

Tub

Merge

Elongation

HA
MORN1

Tub

Inset

Co

Co

MORN1

Tub

Merge

b

RCC1-2-mAID-HA<sub>3</sub>

MORN1

Tub

-IAA

+IAA

MORN1

Tub

c

<figure><img src="image_116.png" /></figure>
**Supplementary Figure 6. RCC1-2 depletion leads to misshapen basal complex. a,** Representative U-ExM images of RCC1-2-mAID-HA₃ upon later endodyogeny, left untreated or IAA-treated for 24h. Intracellular tachyzoites were labeled with anti-HA (green), anti-MORN1 (HA) and anti-α/β tubulin (white) antibodies to label RCC1-2, the basal complex and microtubules, respectively. Nuclear DNA was labeled with Hoechst (blue). Insets highlight developing daughter cells inside mother cell. Images represent maximum-intensity projections of z-stack confocal sections. White arrowheads point developing daughter cells structures (D: daughter) (Ce: centrioles; S: spindle; Co: conoid). Yellow arrowheads mark the rafts of nascents SPMTs. Scale bars are 5µM. RCC1-2 depletion leads to misshapen basal complex.

**b,** Same as **a**, but showing widefield views with basal complex (red) and microtubules (white) labelling. Scale bars are 5µM.

**c,** Representative electron micrographs of dividing RCC1-2-depleted tachyzoites. Left panel, with an inset zoom on one basal end, highlights the basal complex of the daughter cells IMC (white arrowhead). Right panel: Image segmentation shows the IMC in pink, the nucleus in blue, and conoid in yellow. The white arrow indicates a daughter IMC break. Scale bars are 1µm.
<figure><img src="image_117.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_118.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_119.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_120.png" /></figure>

<table><caption>e</caption><thead><tr><th></th><th>212780</th><th>312590</th><th>244500</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>/</td><td>3035bp</td><td>3042bp</td></tr><tr><td>2: P1+P2</td><td>1242bp</td><td>523bp</td><td>384bp</td></tr><tr><td>3: P3+P4</td><td>/</td><td>758bp</td><td>815bp</td></tr><tr><td>Ctrl<br>P1+P4</td><td>/</td><td>427bp</td><td>381bp</td></tr></tbody></table>

<figure><img src="image_121.png" /><figcaption>+J/A</figcaption></figure>
**Supplementary Figure 7. Identification of new daughter-specific proteins by RCC1-2 proximity labelling.** **a,** Illustration of the strategy employed to obtain the RCC1-2-HA₃-TurboID strain using CRISPR/Cas9. Orange arrows depicted the designed primers used to validate the successful integration at the endogenous loci with the expected PCR product sizes.

**b,** Validation of successful integration of the HA₃-TurboID cassette into *TgRCC1-2 (TGGT1_280770)* locus by PCR. The parental strain TaTi served as a negative control (Ctrl).

**c,** Western blot analysis of clear lysates (CL) and eluted proteins (E) from RCC1-2-HA₃-TurboID parasites untreated or treated with biotin for 2 hours. Biotinylated proteins from soluble and cytoskeletal fractions were isolated with anti-streptavidin beads. Samples were immunoblotted with anti-HA (upper panel), anti-streptavidin-HRP (middle panel). TgROP5 was used as loading control (lower panel). RCC1-2 was detected only in the cytoskeletal fraction. **d,** Validation of successful integration of the mAID-HA₃ cassette (-mAID for short) into *TGGT1_212780, TGGT1_312590, TGGT1_244500* locus by PCR. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected PCR product sizes for each primer pair.

**e,** Confocal immunofluorescence images of the three RCC1-2 proximity candidates fused with a mAID-HA cassette (-mAID for short) in IAA-treated parasites for 24 hours. Parasites were stained with anti-HA (green) and anti-IMC1 (magenta) antibodies to visualize HA-tagged proteins and the IMC, respectively. Scale bars are 2µM.
a

<figure><img src="image_122.png" /></figure>

b

<figure><img src="image_123.png" /></figure>

<figure><img src="image_124.png" /></figure>

<table><thead><tr><th></th><th>PCR1</th><th>CPH1</th><th>APR3</th><th>APR2</th><th>KinA</th><th>APR7</th><th>RCC1-2</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>4774bp</td><td>/</td><td>5573bp</td><td>4788bp</td><td>4758bp</td><td>4724bp</td><td>5026bp</td></tr><tr><td>2: P1+P2</td><td>239bp</td><td>3452bp</td><td>1015bp</td><td>231bp</td><td>191bp</td><td>226bp</td><td>466bp</td></tr><tr><td>3: P3+P4</td><td>964bp</td><td>/</td><td>987bp</td><td>986bp</td><td>996bp</td><td>927bp</td><td>989bp</td></tr><tr><td>Ctrl P1+P4</td><td>400bp</td><td>/</td><td>1153bp</td><td>307bp</td><td>438bp</td><td>244bp</td><td>663bp</td></tr></tbody></table>

c

<figure><img src="image_125.png" /></figure>

<figure><img src="image_126.png" /></figure>

d

<figure><img src="image_127.png" /></figure>
<figure><img src="image_128.png" /></figure>

<figure><img src="image_129.png" /></figure>

<figure><img src="image_130.png" /></figure>

<figure><img src="image_131.png" /></figure>

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

<figure><img src="image_134.png" /></figure>

<figure><img src="image_135.png" /></figure>

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /><figcaption>APR8-mAID-HA3 + CPH1-Myc</figcaption></figure>

<table><thead><tr><th></th><th>PCR1</th><th>APR3</th><th>APR2</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>4774bp</td><td>5573bp</td><td>4788bp</td></tr><tr><td>2: P1+P2</td><td>239bp</td><td>1015bp</td><td>231bp</td></tr><tr><td>Ctrl<br>P1+P4</td><td>400bp</td><td>1153bp</td><td>307bp</td></tr></tbody></table>

<figure><img src="image_138.png" /></figure>

Lanes:
1: + PCR1-Ty2
2: + APR2-Ty2
3: + APR3-Ty2
**Supplementary Figure 8.** Generation of double and triple-tagged strains in *TGGT1_212780-mAID-HA<sub>3</sub>* strain. **a**, Confocal immunofluorescence images of *TGGT1_212780-mAID-HA<sub>3</sub>* localization. Parasites were labeled with anti-HA (green) and anti-IMC1 (magenta) antibodies to visualize *TGGT1_212780* and the IMC, respectively. DNA is labeled with Hoechst. White arrowheads point at *TGGT1_212780* signal. Shown are maximum-intensity projections of z-stack confocal images. Scale bars are 2µM. **b**, Validation of successful integration of the MYC2-DHFR cassette into *TgCPH1* locus and TY2-DHFR cassette into *TgPCR1*, *TgAPR3*, *TgAPR2*, *TgKinA*, *TgAPR7* and *TgRCC1-2* locus by PCR within the *TGGT1_212780-mAID-HA<sub>3</sub>* background. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected PCR product sizes for each primer pair. **c**, Assessment of protein expression and depletion of the *TGGT1_212780-mAID-HA<sub>3</sub>* co-expressing either Ty-tagged or Myc-tagged apical complex proteins. Immunoblot analysis using an anti-HA, anti-Ty or anti-Myc antibodies was performed on lysates from intracellular parasites that were either left untreated or treated with IAA for 6h. TgROP5 was used as loading control. Successful insertional tagging of CPH1-Myc2, PCR1-Ty2, APR3-Ty2, APR2-Ty2, KinA-Ty2, APR7-Ty2 and RCC1-2-Ty2 was verified and their expression levels remained unchanged upon RCC1-2 depletion. Protein molecular weights (in kDa) are indicated on the left of each panel. **d**, U-ExM images of *TGGT1_212780-mAID-HA<sub>3</sub>* in combination with multiple Ty/Myc-tagged apical complex proteins, shown as top view of a single daughter cell. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty or anti-Myc (green) and anti-α/β tubulin (white) antibodies to label *TGGT1_212780*, apical markers and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. Scale bars are 2µM. **e**, Colocalization of APR8-mAID-HA3 with Ty-tagged proteins KinA, APR7 and RCC1-2 by U-ExM. Intracellular tachyzoites were stained with anti-HA (red), anti-Ty (green) and anti-α/β tubulin (white) antibodies to label APR8, Ty-tagged proteins and
microtubules, respectively. The direction used to measure the fluorescence intensity in daughter
cells, which generates the colocalization curve, is indicated by the yellow bar. Scale bars are
1µM. **f**, U-ExM images of APR8-mAID-HA₃ in combination with the Myc-tagged protein
SFA2. Intracellular tachyzoites were stained with anti-HA (red), anti-Myc (green) antibodies
to label APR8 and the striated fiber component SFA2, respectively. Nuclear DNA was labeled
with Hoechst (blue). Images represent maximum-intensity projections of z-stack confocal
sections. Scale bars are 1µM. **g**, Validation of successful integration of the TY2-DHFR cassette
into *TgPCR1*, *TgAPR3* and *TgAPR2* locus by PCR into the APR8-mAID-HA₃ + CPH1-My2
background. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected
PCR product sizes for each primer pair. **h**, Validation of successful expression of PCR1-Ty2,
APR3-Ty2 and APR2-Ty2 in APR8-mAID-HA₃ + CPH1-Myc2 background. Immunoblot
analysis using an anti-HA, anti-Ty and anti-Myc antibodies was performed on lysates from
intracellular parasites. TgROP5 was used as loading control. Protein molecular weights (in kDa)
are indicated on the left of each panel.
a

APR8-mAID-HA<sub>3</sub> +
CPH1-Myc

<figure><img src="image_139.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
PCR1-Ty

<figure><img src="image_140.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
APR3-Ty

<figure><img src="image_141.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
APR2-Ty

<figure><img src="image_142.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
KinA-Ty

<figure><img src="image_143.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
APR7-Ty

<figure><img src="image_144.png" /></figure>

APR8-mAID-HA<sub>3</sub> +
RCC1-2-Ty

<figure><img src="image_145.png" /></figure>

b

<figure><img src="image_146.png" /></figure>

<table><thead><tr><th></th><th>APR8</th></tr></thead><tbody><tr><td>1: P1+P4</td><td>4873bp</td></tr><tr><td>2: P1+P2</td><td>367bp</td></tr><tr><td>3: P3+P4</td><td>935bp</td></tr><tr><td>Ctrl<br>P1+P4</td><td>392bp</td></tr></tbody></table>

c

<figure><img src="image_147.png" /></figure>

d

Initiation

APR8-mAID-HA<sub>3</sub> + CPH1-Myc

<figure><img src="image_148.png" /></figure>

Nucleation

Elongation

e

<figure><img src="image_149.png" /></figure>

f

<figure><img src="image_150.png" /></figure>
**Supplementary Figure 9. APR8 depletion impairs APR organization.** **a,** Representative U-ExM images of mother apical complex in APR8-mAID-HA₃ strain with Ty-/Myc-tagged apical markers, either left untreated or IAA-treated for 6h. Intracellular tachyzoites were stained with anti-Ty or anti-Myc (green) and anti-α/β tubulin (magenta) antibodies to label apical markers and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections, with inset zoom highlighting a single mother apical complex. White arrowheads point mother conoid. Scale bars are 1µM. **b,** Validation of successful integration of the TY2-DHFR cassette into *TgAPR8* locus by PCR into the RCC1-2-mAID-HA₃ background. The parental strain Tir1 served as a negative control (Ctrl). Table lists expected PCR product sizes for each primer pair. **c,** Assessment of protein expression and depletion of the RCC1-2-mAID-HA₃ co-expressing Ty-tagged APR8. Immunoblot analysis using an anti-HA and anti-Ty antibodies was performed on lysates from intracellular parasites that were either left untreated or treated with IAA for 24h. Validation of successful expression of APR8-Ty2 in RCC1-2-mAID-HA₃ background. Immunoblot analysis using an anti-HA and anti-Ty antibodies was performed on lysates from intracellular parasites. TgROP5 was used as loading control. Protein molecular weights (in kDa) are indicated on the left of each panel. **d,** U-ExM images of APR8-mAID-HA₃ + CPH1-Myc2 from initiation to elongation stage of endodyogeny. Intracellular parasites were either left untreated or IAA-treated for 4h. Parasites were stained with anti-Myc (green) and anti-α/β tubulin (magenta) antibodies to label the conoid protein CPH1 and microtubules, respectively. Images represent maximum-intensity projections of z-stack confocal sections. Insets highlight developing daughter cells. White arrowheads point developing daughter cells structures (Ce: centrioles; S: spindle; Co: conoid). Scale bars are 1µM. **e,** Representative transmission electron microscopy (TEM) images of APR8-depleted tachyzoites. Black arrows highlight nascent daughter conoid (dCo) is still connected to centrioles (ce) via the striated fiber (sfa). (dIMC: daughter IMC; cc: centrocone;
daughter IMC; cc: centrocone; N: nucleus). Scale bars are 0,5µm. f, Quantification of SFA labeling by indirect immunofluorescence analysis in APR8-mAID-HA3 parasites, left untreated or IAA-treated for 6 hours. Vacuoles were quantified from approximately 100 vacuoles and are presented as mean ± SD (n = 3 biological replicates). Statistical significance was determined by unpaired two-tailed Student's t-test.
**Supplementary movie 1: Imaris 3D reconstruction of U-ExM images from intracellular RCC1-2-mAID-HA₃ parasites.** Parasites were stained with anti-HA (green) and anti-α/β tubulin (magenta) antibodies to label RCC1-2 and microtubules, respectively. Nuclear DNA is labelled with Hoechst. RCC1-2 and microtubules were segmented using the Surface module. Scale bars are 10µM. Synchronous division occurs in rosette-patterned parasitophorous vacuoles and RCC1-2 localized at the apical end of developing daughter cells.

**Supplementary movie 2: Imaris 3D reconstruction of U-ExM images from intracellular RCC1-2-mAID-HA₃ parasites upon IAA depletion.** Parasites were stained with anti-HA (green) and anti-α/β tubulin (magenta) antibodies to label RCC1-2 and microtubules, respectively. Nuclear DNA is labelled with Hoechst. Microtubules were segmented using the Surface module. Scale bars are 10µM. Tachyzoites undergo asynchronous division and presents SPMTs defects.

**Supplementary movie 3: Imaris 3D reconstruction of U-ExM images from extracellular RCC1-2-mAID-HA₃ parasites.** Parasites were stained anti-α/β tubulin (magenta) antibodies and Hoechst to label microtubules and nuclear DNA, respectively. Microtubules were segmented using the Surface module. Scale bars are 5µM. Tachyzoites appears as elongated crescent shape with 22 SPMTs anchored at the APR.

**Supplementary movie 4: Imaris 3D reconstruction of U-ExM images from extracellular RCC1-2-mAID-HA₃ parasites upon IAA depletion.** Parasites were stained anti-α/β tubulin (magenta) antibodies and Hoechst to label microtubules and nuclear DNA, respectively. Microtubules were segmented using the Surface module. Scale bars are 5µM. The symmetry
and organization of SPMTs in tachyzoites is disrupted in absence of RCC1-2. Parasites lacked the full complement of 22 SPMTs and lost their elongated crescent shape.

**Supplementary movie 5: Imaris 3D reconstruction of U-ExM images from intracellular RCC1-2-mAID-HA₃ parasites with basal complex labelling.** Parasites were stained with anti-HA (green), anti MORN1 (red) and anti-α/β tubulin (white) antibodies to label RCC1-2, the basal complex and microtubules, respectively. Nuclear DNA is labelled with Hoechst. RCC1-2, basal complex and microtubules were segmented using the Surface module. Only one daughter cell was 3D-reconstructed to facilitate visualization of the connection between the basal complex and the SPMTs. Scale bars are 5µM. RCC1-2 localized to the apical end of daughter cell. MORN1 localized to the basal end of daughter cells as a ring structure connected to daughter SPMTs. Daughter cells form elongated domes within the mother cell

**Supplementary movie 6: Imaris 3D reconstruction of U-ExM images from intracellular RCC1-2-mAID-HA3 parasites with basal complex labelling upon IAA depletion.** Parasites were stained with anti-HA (green), anti MORN1 (red) and anti-α/β tubulin (white) antibodies to label RCC1-2, the basal complex and microtubules, respectively. Nuclear DNA is labelled with Hoechst. RCC1-2, basal complex and microtubules were segmented using the Surface module. Only one replicating mother cell was fully 3D-reconstructed to facilitate visualization of the SPMTs defects. Scale bars are 5µM. Daughter cells show flattened morphology upon RCC1-2 depletion, concomitant with SPMT shape defects. The corresponding ring structure observed for the basal complex is often irregular or appeared as discontinuous segments.