PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation ofPlasmodium falciparum

9 Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in 10 eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain 11 the structural integrity and loading of this complex on mitotic chromosomes. This complex is 12 present in all eukaryotes and has recently been identified in Plasmodium spp. However, how 13 this complex is assembled and whether the kleisin subunit is critical for this complex in these 14 parasites is yet to be explored . To examine the role of PfCAP -H during cell division within 15 erythrocytes, we generated an inducible PfCAP -H knockout parasite. We find that PfCAP-H is 16 dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-17 H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the 18 absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the 19 mitotic chromosomes. While mitotic spindle assembly is intact i n PfCAP-H deficient parasites, 20 duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed 21 karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis 22 occurs normally. Altogether, our data suggest that PfCAP -H plays a crucial role in maintaining 23 the structural integrity of the condensin I complex on the mitotic chromosomes and is essential 24 for the asexual development of malarial parasites. 25 26 Importance 27 Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their 28 survival within two distinct hosts - human and Anopheles mosquitoes. Despite its great 29 significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, 30 mitosis is regulated by one of the pivotal complexes known as condensin complexes . The 31 condensin complexes are responsible for chromosome condensation , ensuring the faithful 32 distribution of genetic material to daughter cells. While condensin complexes have recently been 33 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint identified in Plasmodium spp, our understanding of how this complex is assembled and their 34 precise functions during the blood stage development of Plasmodium falciparum remains largely 35 unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood 36 stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a 37 pivotal role in upholding the structure of condensin I and facilitating karyokinesis. 38 39

Plasmodium, mitosis, chromosome, condensin I, karyokinesis 40 41

42 Plasmodium falciparum is a protozoan parasite that is responsible for the most severe forms of 43 human malaria. Malaria remains one of the most important global infectious diseases, claiming 44 more than 619 000 lives worldwide annually [1]. These parasites have a complex life cycle that 45 alternates between two different hosts, Anopheles mosquitoes and humans. To survive and 46 thrive in these hosts, they replicate through an atypical cell division . These parasites follow 47 schizogony within hepatocytes and red blood cells (erythrocytes) in their human hosts. On the 48 other hand, they undergo gametogenesis and sporogony in mosquitoes [2]. Among all these 49 stages, the extensive proliferation of parasites within human erythrocytes causes the signs and 50 symptoms of clinical malaria. Parasite cell division in erythrocytes, known as schizogony, is an 51 unconventional mode of cell division that includes growth and budding phases [3, 4]. During the 52 growth phase, parasite undergo several asynchronous rounds of DNA replication and mitosis 53 (S–M phase) without cytokinesis [5, 6]. Asexual Plasmodium parasites undergo closed mitosis, 54 where decondensed chromosomes are segregated , followed by nuclear division with an intact 55 nuclear envelope throughout the cycle [2]. Later in the budding phase, the parasite undergoes 56 a final round semi-synchronous nuclear division along with cytokinesis to produce mature 57 daughter cells, called merozoites . These merozoites invade new erythrocytes to begin the 58 proliferation cycle again [7, 8] . Despite its predominant role in asexual proliferation, the 59 regulation of this atypical mitosis is still underexplored in Plasmodium parasites. 60 61 During mitosis, chromatin condensation and segregation are important events to ensure that 62 genomic material is equally divided into the daughter nuclei [9, 10]. This process is mediated by 63 two distinct condensin complexes , condensin I and II . These complexes are pentameric and 64 comprised of two parts – core subunits and regulatory subunits. The core subunit consists of 65 structural maintenance of chromosomes (SMC) 2 and 4, common in condensin I and II 66 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint complexes. In contrast, the regulatory subunits, collectively known as chromosome-associated 67 proteins (CAP) or non-SMC members, differ between two complexes; condensin I contains CAP-68 H/CAP-G/CAP-D2 while condensin II has CAP-H2/CAP-G2/CAP-D3 [11, 12]. While condensin I 69 is highly conserved across eukaryotes and its localization is dynamic throughout the cell division, 70 condensin II remains in nucleus and is absent in some organisms (e.g., yeast and insects) [12, 71 13]. 72 73 In Plasmodium sp p., both condensin I and II complex have been identified by homology 74 prediction [14, 15]. The two core subunits (SMC 2/4) and one non -SMC member- PfCAP-G of 75 the condensin I complex have been genetically interrogated in Plasmodium spp. [14, 16]. In 76 Plasmodium berghei, SMC2/4 displayed a dynamic localization in both asexual parasites and 77 gametocytes. PbSMC2/4 knockout in asexual parasites was unsuccessful, therefore a functional 78 evaluation was not possible during that stage. Depletion of PbSMC2/4 during 79 gametocytogenesis impaired male gametogenesis and zygote differentiation and thus blocked 80 parasites transmission in P. berghei [14]. 81 82 On the other hand, the knockdown of PfCAP-G (or Merozoite Organizing Protein - MOP), a non-83 SMC member of the condensin I complex, showed a fitness defect in the asexual development 84 of P. falciparum. The PfCAP-G-deficient parasites showed flawed segmentation with a large 85 residual agglomerate of partially divided cells [16]. Given that the PfCAP -G knockdown 86 phenotype was incomplete, likely due to insufficient protein knockdown , the phenotype of 87 complete loss of condensin I remains insufficiently evaluated. Furthermore, there remains a lack 88 of experimental evidence on how these complexes are assembled on chromosomes and their 89 function as regulators of mitosis during the asexual blood stage development in P. falciparum. 90 91 In eukaryotes, the kleisin subunit CAP-H acts as a linchpin in the assembly of the condensin I 92 complex [17]. The CAP-H sequence consists of five motifs that bind to different components of 93 the condensin I complex [18-20]. The N- and C- terminal motifs of this protein interact with the 94 core proteins (SMC 2 and SMC 4) , while the central regions consist of motifs that bind to two 95 other non -SMC proteins - CAP-G and CAP -D2 along with a region that interacts with 96 chromosomal DNA to anchor condensin [18, 21-23]. In addition, the loading of the condensin I 97 complex during mitosis is regulated by the N -terminal tail of CAP -H in Xenopus egg extracts 98 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint [24]. Mutation or deletion of CAP -H results in a mitotic chromosome condensation and 99 segregation defect in yeast and Drosophila melanogaster [25-27]. 100 101 CAP-H has been bioinformatically predicted in Plasmodium spp. [14], and we have focused on 102 interrogating its function during the asexual development of Plasmodium falciparum within 103 human erythrocytes. We generated a parasite strain allowing inducible knockout of PfCAP-H. 104 We show that PfCAP-H is a member of the condensin I complex and is essential for the asexual 105 parasite replication. PfCAP-H is dynamically localized during mitosis and can be used as a 106 marker for the metaphase plate. Depletion of PfCAP -H causes abnormal karyokinesis , while 107 cytokinesis occurs normally. This study provides new insights into the function of the condensin 108 I complex during asexual replication of P. falciparum. 109 110

111 112 PfCAP-H has conserved N- and C- terminal region and is expressed in proliferative blood 113 stages 114 CAP-H is a central component of the condensin I complex in all eukaryotes [17]. Bioinformatic 115 analysis predicted that CAP-H is also present in Plasmodium falciparum [14]. PF3D7_1304000 116 (hereafter referred to as PfCAP-H) is 1024 amino acids long with a putative condensin complex 117 subunit 2 domain. To comprehensively evaluate the PfCAP-H sequence by in silico analysis, we 118 compared the sequence of PfCAP -H with CAP-H homologs from a wide range of eukaryotes. 119 PfCAP-H consists of conserved regions in its N (1 – 250 aa) and C (890 – 1000 aa) termini, with 120 a less conserved interior sequence (Fig. S1A). The complete amino acid sequence has 21-34% 121 similarity with the majority of the non-Apicomplexan homologs and, as expected, higher similarity 122 to Plasmodium homologs (e.g., 64% to PbCAP-H) (Fig. S1B). 123 124 To directly investigate the role of PfCAP-H in Plasmodium falciparum, we generated an inducible 125 PfCAP-H knockout (iKO) strain in 3D7pfs47DiCre parasites (named PfCAP-HDiCre) [28] (Fig. 1A). 126 In these parasites , the native PfCAP -H gene locus has been replaced with a loxP-flanked, 127 codon-altered PfCAP -H with the spaghetti monster ( sm)V5 epitope tag at the C -terminus. 128 Integration of the donor cassette was verified by PCR amplification. The expected size of 2.3 129 and 0.98 kilobase-pairs (kb) was observed in the transgenic line, while no equivalent bands were 130 amplified in the parental line (Fig. S2A). 131 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint 3'UTRhDHFR5'UTR caPfCAP-H 3'UTR5'UTR smV5 smV5 caPfCAP-H hDHFR Rapa Donorcassette PfCAP-HDiCre Pf3D7DiCre ΔPfCAP-HDiCre 3'UTRPfCAP-H5'UTR 1 4 loxP DiCre 3'UTR5'UTR hDHFR A C B 3D7 DiCre PfCAP-H DiCre PfCAP-H150 kDa 15 kDa R T S H3 Merge PfCAP-HCentrin .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 1: Expression and localization of PfCAP -H in PfCAP -HDiCre parasites. (A) Schematic of 132 PfCAP-HDiCre parasites. In PfCAP -HDiCre parasites, we replaced the endogenous locus of PfCAP-H in 133 3D7pfs47DiCre (referred to as 3D7DiCre) parasites with a loxP-flanked (triangle), codon-altered PfCAP-H 134 with spaghetti monster (sm)-V5 tag (magenta-colored box) at the C-terminus. (B) Immunoblot showing 135 the expression of PfCAP -H during asexual blood stages (R - Rings, T - Trophozoites, S - Schizonts) 136 probed with α-V5. The parental Pf3D7DiCre parasites are used as a negative control and α-Histone H3 was 137 used as a protein loading control. (C) The localization of PfCAP-H was visualized by α-V5 (magenta) for 138 smV5 tagged PfCAP-H and α-Centrin (green) was used as a marker of centrosome by slide-based IFA. 139 The IFA showed that PfCAP-H is localized near centrosome. The DNA was stained with Hoechst 33342 140 (blue). Scale bar = 2 µm. 141 142 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint We next sought to determine the expression timing and localization of PfCAP -H during the 143 asexual blood stages. The immunoblot, probed with a-V5, demonstrated the expected 150 kDa 144 of smV5-tagged PfCAP-H in both trophozoites and schizonts (Fig. 1B). The observed PfCAP-H 145 expression agrees with the transcriptional profiling data [15], indicating a likely role for PfCAP-H 146 during asexual development. Given that PfCAP-H homologs are present near centrosome s in 147 other organisms [12], we performed an immunofluorescence assay (IFA) on schizonts with an 148 antibody that recognizes Plasmodium centrins as a marker for the centrosome in these 149 parasites. PfCAP -H localize s near the centrosomes (Fig. 1C), similar to the reported 150 localizations for PbSMC2/4 [14]. 151 152 PfCAP-H is highly expressed in mitotically active nuclei during schizogony 153 To interrogate the subcellular localization of PfCAP-H during the proliferative stages of asexual 154 development, we performed IFAs throughout the schizont stage of the asexual development 155 cycle, probing for PfCAP-H (a-V5) together with the nuclear (DNA) stain Hoechst 33342. The 156 IFA revealed discrete perinuclear foci throughout schizogony (Fig. S3), with diminished protein 157 detection when segmentation is complete . This expression resembles the pattern of proteins 158 crucial for cell cycle progression [29]. 159 160 For higher resolution imaging, we used u ltrastructure-expansion microscopy (U-ExM) to more 161 precisely localize PfCAP-H and examine its role during cell division in these parasites. For these 162 studies, we have included a fluorophore conjugated to N-hydroxysuccinimide (NHS) (herein 163 referred to as “NHS ester”) as a non-specific stain for protein density [30], SYTOX Deep Red as 164 a DNA stain, a-V5 for smV5-tagged PfCAP-H, and a-tubulin for mitotic spindles. As noted above, 165 P. falciparum undergoes an atypical cell division known as schizogony, where individual nuclei 166 undergo asynchronous S/M cycles, followed by a final semi-synchronous cycle coupled with the 167 budding of the daughter cells [4, 29]. Given the complexity of schizogony and decondensed 168 chromosomes in Plasmodium falciparum, it is challenging to distinctly visualize different stages 169 of mitosis in these parasites. Therefore, we relied upon the positioning of mitotic 170 apparatus/microtubule organizing centers and microtubules to evaluate the distinct phases of 171 mitosis in these parasites. As shown in the Fig. 2A, in nuclei undergoing mitosis, we observed 172 that PfCAP-H shows a speckled pattern at the plus end tip of the mitotic spindles. The signal 173 intensifies and organizes as clusters at the metaphase plate where chromosomes are typically 174 aligned during metaphase. Subsequently, these signals resume their speckled pattern at the tip 175 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A Prophase Anaphase Telophase Metaphase Merge w/SYTOX Merge w/NHS-Ester PfCAP-H w/ Tubulin B Merge w/ SYTOX Merge w/ NHS-Ester PfCAP-H w/ Tubulin .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 2: PfCAP-H is present in mitotically active nuclei. (A) PfCAP-HDiCre parasites were prepared 176 for U-ExM and stained with α-V5 for PfCAP-H (in magenta), α-tubulin for mitotic spindles (in green), 177 NHS-Ester for protein stain (in grayscale), and SYTOX for nuclear stain (in cyan) and captured using 178 Airyscan microscopy. With the cues from the localization of mitotic spindles, different stages of mitosis 179 (prophase, metaphase, anaphase, and telophase) are marked compared to position of mitotic spindles 180 in conventional mitotic stages. The peak expression of PfCAP-H is at the metaphase plate. Scale bars 181 = 2 µm. (B) Example of schizont exhibiting two mitotic spindles within one dividing nuclei and thus 182 suggesting the existence of schizogony with limited karyokinesis during blood stages. Scale bars = 2 183 µm, image is projection of 20 z-slices. 184 185 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint of mitotic spindles during anaphase and telophase, which finally becomes more diffuse at the 186 end of the cell cycle. To further confirm the localization of PfCAP -H to the plus end of mitotic 187 spindles, we generated a parasite strain where the kinetochore marker, 188 PfNDC80/PF3D7_0616200[31, 32] , was additionally epitope -tagged at its endogenous locus 189 with spaghetti monster HA (smHA) ( Fig. S4A and S4B) . Immunofluorescence of these dual -190 tagged parasites demonstrated that PfCAP -H localizes within the surrounding PfNDC80 191 staining, suggesting that PfCAP-H is on the chromosomal side ( Fig. S4C). These results 192 demonstrate that PfCAP -H shows a dynamic pattern during different stages of mitosis and 193 provides a marker for the metaphase plate in mitotically active parasites. 194 195 Interestingly, we observed that, in ~10% of schizonts (3 of 31), two duplicated hemi -spindle 196 and/or mitotic spindle s are present within a single nucleus (Fig. 2B). These unusual events 197 suggest that these schizonts follow unconventional schizogony where already duplicated 198 genomes with a single, undivided nucleus have already started the next round of mitosis before 199 completing karyokinesis. This supports a model of schizogony with limited karyokinesis [4] 200 occurring at times during asexual development. 201 202 PfCAP-H is a member of the condensin I complex in Plasmodium falciparum 203 The observed expression and localization pattern of PfCAP -H resembles the pattern exhibited 204 by SMC core members ( PbSMC2/4) and PfCAP-G of the condensin I complex in Plasmodium 205 spp. [14, 16] , suggesting that PfCAP -H is a member of the condensin I complex in these 206 parasites. To confirm this hypothesis in Plasmodium falciparum, we performed two independent 207 experiments. First, we investigated whether PfCAP -H interacts with PfCAP -G, a non -SMC 208 member of the condensin I complex by performing IFA with PfCAP-HDiCre /PfCAP-G parasites, 209 where the endogenous PfCAP-G has a smHA epitope in the smV5-tagged PfCAP-H strain. The 210 dual-transgenic line was confirmed by integration PCR and whole genome sequencing (Fig. 211 S5A-S5C, sequence reads deposited in NCBI Sequence Read Archive, #XXXXXX ). PfCAP-H 212 and PfCAP-G colocalize with each other and displayed a similar dynamic expression pattern 213 throughout schizogony (Fig. 3A and S5D), suggesting that these two proteins interact with each 214 other throughout this stage . Second, we used an alternative approach where we fused a 215 promiscuous version of the biotin ligase BirA[33] to PfCAP-G in 3D7 parasites to generate a new 216 transgenic PfCAP-GBirA strain. We used this PfCAP-GBirA parasites to identify the proteins that 217 interact with PfCAP-G using BioID-based proximity labeling in late schizonts coupled with mass 218 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A B PfCAP-H PfCAP-G Merge 0.0 0.1 0.2 0.3 0.4 0 20 40 60 80 Fractional Coverage # of Unique Peptides PfCAP-G PfSMC2 PfCAP-H PfSMC4PfCAP-D2 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 3: PfCAP-H is a member of the condensin I complex. (A) Schizonts from PfCAP-HDiCre/PfCAP-219 G parasites were fixed and stained with α -V5 against PfCAP-H and α-HA against PfCAP-G (n= 3). The 220 slide-based IFA showed that PfCAP -H colocalizes with PfCAP -G. Scale bars = 2 µm (B) Proximity 221 labeling in PfCAP-GBirA parasites demonstrates that PfCAP-H interacts with PfCAP-G and other members 222 of the condensin I complex (representative example shown from one of three biological replicates). 223 224 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint spectrometry (Fig. 3B and Supplemental Table S1) [33]. As anticipated, all the members of the 225 condensin I complex including PfCAP-H were among the top hits, implying that PfCAP -G is a 226 true partner of PfCAP-H along with other members of the condensin I complex. Altogether, these 227 data establish PfCAP-H as a bona fide member of the condensin I complex in P. falciparum. 228 229 PfCAP-H is essential for asexual development of blood stage parasites 230 The condensin I complex plays a crucial role during cell division. Knockdown of PfCAP -G 231 produces a significant fitness defect in P. falciparum, and PbSMC2/4 could not be knocked out 232 in P. berghei asexual parasites[14, 16]. To directly determine the consequences for complete 233 removal of PfCAP-H, we performed a replication growth assay where we treated synchronized 234 early ring (0-4 hrs) staged PfCAP-HDiCre parasites with rapamycin or DMSO and monitored their 235 growth for two consecutive cycles using flow cytometry. In these parasites (Fig. 1A), rapamycin 236 dimerization of the DiCre subunits mediates excision of the PfCAP-H gene flanked by two loxP 237 sites, resulting in a PfCAP-H knockout ( PfCAP-H iKO) [28]. In PfCAP-H iKO parasites, whole 238 locus PCR amplification detected 4.8 kb truncated gene locus in rapamycin treated parasites 239 compared to full length 9.5 kb modified PfCAP-H locus in DMSO-treated parasites (Fig. S2A). 240 Furthermore, IFA demonstrated that the rapamycin treatment resulted in complete loss of 241 PfCAP-H protein in PfCAP -H iKO parasites (Fig. S2B) We used t he parental 3D7 pfs47DiCre 242 parasite strain as a control. The rapamycin-treated PfCAP-HDiCre parasites exhibited a 97.8% ± 243 0.6 growth defect resulting in death of parasites within the same cycle of treatment when 244 compared to DMSO-treated parasites (Fig. 4A). 245 246 To evaluate the growth phenotype in more detail, we examined field -stained parasites to 247 determine the developmental stage of the arrested parasites . At this level of resolution, w e 248 observed that PfCAP -H iKO parasites grew normally until the early schizont stage 249 (approximately 30 hours post -invasion [hpi]). After this point, parasites had abnormal 250 morphology and, eventually, failed to generate newly invaded rings (Fig. 4B). Because we did 251 not observe an accumulation of unruptured schizonts, we measured the timing of egress (and 252 potential reinvasion or lack thereof), in the presence and absence of PfCAP-H knockout, by flow 253 cytometry. Notably, we did not find any significant difference in the timing of schizont egress in 254 the absence of PfCAP-H. PfCAP-H iKO parasites ruptured at the same time as control parasites, 255 suggesting that loss of PfCAP-H does not cause an egress defect . However, the egressed 256 PfCAP-H iKO parasites did not form new rings (Fig. 4C). 257 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A B C 40 45 50 55 60 0.0 0.2 0.4 0.6Parasitemia(%) Schizonts hours post invasion (hpi) 40 45 50 55 60 0 2 4 6Parasitemia(%) Rings hours post invasion (hpi) PfCAP-H DiCre + DMSO PfCAP-H DiCre + Rapa PfCAP-H DiCre + DMSO PfCAP-H DiCre + Rapa 0 2 4 0 2 4 6 8 10 Days Parasitemia(%) 0 - 4 26 - 30 34 - 38 44 - 48 50 - 54hpi Rapa DMSO .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 4: PfCAP-H is essential for asexual development of parasites. (A) PfCAP-HDiCre parasites 258 were treated with rapamycin and their growth was monitored over two consecutive cycles by flow 259 cytometry. Loss of PfCAP-H showed a lethal effect on parasite asexual growth development. Error bars 260 show standard deviation from mean of three independent biological replicates. (B) The Hemacolor 261 stained smear was prepared for the first growth cycle to observe progression of asexual stages. In 262 rapamycin treated parasites, we observed that the parasites are arrested at the late blood stages and no 263 new invasion of red blood cells were observed. (C) Time course of PfCAP -H parasites cultured with 264 rapamycin/DMSO were collected every 5 hours from 40 to 60 hpi, and parasitemia was measured by flow 265 cytometry (n = 3, error = standard error of mean (SEM)) to examine the duration of egress and invasion. 266 Rapamycin- treated parasites did not show any significant difference in the progression of growth, 267 however they could not invade new red blood cells. 268 269 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint We next asked whether this growth defect is due to alterations in DNA replication in PfCAP -H 270 iKO parasites. To address this, we measured the DNA content of rapamycin or DMSO treated 271 parasites at 40 hpi, when parasites are at the peak of their dividing state . Parasite DNA was 272 labeled with SYBR Green and quantified by measurement of mean fluorescence intensity (MFI) 273 by flow cytometry. Remarkably, we did not find any significant difference in the total DNA content 274 in PfCAP-H iKO and control parasites (Fig. S6), indicating that deletion of PfCAP -H does not 275 have a major effect on DNA replication in the knockout parasites. 276 277 PfCAP-H depletion does not affect the centrosome and mitotic spindle formation 278 Centrosomes act as the microtubule -organizing center (MTOC), and failure to duplicate or 279 separate could lead to aberrant cell division [34]. We interrogated whether the depletion of 280 PfCAP-H affects the biogenesis and dynamics of centrosomes during mitosis with U-ExM on the 281 DMSO and rapamycin-treated PfCAP-HDiCre strains probed with a-V5, a-Centrin, NHS-Ester, 282 and SYTOX [35]. Surprisingly, t he centrosomes are present and duplicated normally, even 283 without PfCAP -H (Fig. 5A and 5B). However, in the absence of PfCAP -H, the centrosomes 284 remain clustered over the mass of unsegmented nuclei . These results suggest that the 285 phenotype observed by depletion of PfCAP-H is not due to failure in centrosome duplication or 286 separation but to some other defect. 287 288 We monitored if mitotic spindles could form in the absence of PfCAP-H by direct visualization of 289 these structures by U-ExM with a-tubulin in dividing parasites. In rapa mycin-treated parasites, 290 we observed normal mitotic spindle formation, suggesting that the PfCAP-H is also not required 291 for mitotic spindle assembly (Fig. 5C). Taken together, these results implies that the PfCAP-H 292 function is dispensable for centrosome and mitotic spindle assembly. 293 294 Loss of PfCAP-H deters the proper localization of PfCAP-G on mitotic chromatin 295 PfCAP-H homologs play a vital role in maintaining the ring-like structure of condensin I complex 296 and ensuring their collective function as chromatin condensers in other eukaryotes [17]. To 297 investigate if this function was preserved for PfCAP-H, we evaluated the expression and 298 localization of PfCAP-G in the presence or absence of PfCAP-H. With U-ExM, we found that the 299 PfCAP-G is no longer localized to the mitotic chromosomes in the PfCAP-H iKO parasites (Fig. 300 6A). However, the expression of PfCAP-G is not affected in the absence of PfCAP-H (Fig. 6B). 301 This observation suggests that the depletion of PfCAP-H does not affect the expression of 302 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A DMSO Rapa PfCAP-H w/ Hoechst Centrin w/ Hoechst Merge Inset B DMSO Rapa PfCAP-H w/ Centrin + NHS-Ester Merge w/ SYTOX DMSO Rapa PfCAP-H w/ Tubulin + NHS-Ester Merge w/ SYTOX C .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 5: PfCAP-H is dispensable for centrosome dynamics and microtubule formation. 303 PfCAP-HDiCre/PfCAP-G parasites were synchronized, treated with rapamycin/DMSO, and 304 collected for slide-based IFA and U-ExM. The samples were stained with α-V5 (PfCAP-H, 305 magenta), α-Centrin (Centrosome marker, green), and α-tubulin (green). In addition, for U-306 ExM, NHS-Ester (greyscale) and SYTOX (cyan) was used. (A) In PfCAP-H deficient parasites, 307 the slide-based IFA demonstrated that centrosome is duplicated but their separation is 308 affected. The highly resolved U-ExM further confirms the similar observation. The U-ExM 309 showed that centrosome (B) and microtubule formation (C) look similar in both the conditions. 310 Scale bar = 2 µm for images and inset. 311 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A B DMSO Merge w/ SYTOXMergePfCAP-G w/ NHS-Ester PfCAP-H w/ NHS-Ester Rapa DMSO Rapa PfCAP-H 150 kDa PfCAP-G 250 kDa α-H3 15 kDa .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 6: PfCAP -H is essential for assembly of condensin I complex. PfCAP-HDiCre/PfCAP-G 312 parasites were synchronized, treated with rapamycin/DMSO, and collected for U-ExM. The samples were 313 stained with α-V5 (PfCAP-H, magenta), and α-HA (PfCAP-G, green), NHS-Ester (greyscale), and SYTOX 314 (cyan). (A) In PfCAP-H deficient parasites, the highly resolved U-ExM showed that localization of PfCAP-315 G to the mitotic chromosome is lost in the absence of PfCAP-H. (B) The immunoblot analysis displayed 316 that in PfCAP-H KO parasites expression of PfCAP-G is unaffected. Scale bar = 2 µm. 317 318 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint PfCAP-G, but it is crucial for the loading of PfCAP-G on the mitotic chromosomes. Thus, the 319 data implies that PfCAP-H is critical for the assembly for non-SMC members of the condensin I 320 complex in Plasmodium falciparum. 321 322 Depletion of PfCAP-H affects karyokinesis but does not impede cytokinesis 323 Our observations thus far suggest that the defect in PfCAP-H iKO parasites is due to abnormal 324 nuclear division rather than DNA replication. We thus interrogated the process of karyokinesis 325 in these parasites during their asexual development. Again, we utilized U-ExM along with NHS-326 Ester, a-V5 for PfCAP-H, and SYTOX to visualize the parasite DNA. At 40 hpi we observed that 327 PfCAP-H iKO parasites showed defective nuclear division . At the same stage, the control 328 parasites had normal nuclear division (Fig. S7). We further followed the karyokinesis until the 329 end of one development cycle by trapping parasites prior to egress with the cysteine protease 330 inhibitor E64 [36]. Strikingly, we observed that fully matured PfCAP -H iKO parasites showed a 331 large agglomerate of incompletely separated nuclear material compared to the >20 normally 332 separated nuclei in control parasites (Fig. 7A). This result suggests that the defect of 333 chromosome segregation leads to a defect in karyokinesis. 334 335 To evaluate cytokinesis, E64-treated parasites were stained with anti bodies agai nst V5 and 336 PfGAP45, an inner membrane complex (IMC) associated protein .[37] With U-ExM, we 337 demonstrated that despite abnormal karyokinesis, the IMC still surrounds the nascent 338 merozoites. A varied amount of nuclear material is contained in the forming merozoites with 339 some nuclear material observed in streaks within the contracted basal complex as well as 340 additional nuclear material in the residual body (Fig. 7B). While there may be abnormalities in 341 the final steps of abscission, these results suggest that the processes of cytokinesis, including 342 IMC formation and basal complex contraction, are largely intact. These schizonts still egress 343 (Fig. 4C) but do not form new ring-stage parasites. 344 345 PfCAP-H is likely dispensable for sexual stage development 346 Given that PbSMC2/4 and PfCAP -G are necessary for the sexual development of these 347 parasites, we asked whether PfCAP -H is required for the sexual development of Plasmodium 348 falciparum. To investigate the role of PfCAP-H in gametocyte development, we induced sexual 349 commitment in synchronized PfCAP-HDiCre parasites, treated with rapamycin or DMSO, and then 350 monitored the gametocytogenesis over 12 days. The gametocyte conversion rate was similar 351 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A B DMSO Rapa PfCAP-H w/ PfCAP-G + SYTOX PfCAP-H w/ PfCAP-G + NHS-Ester Merge DMSO Rapa PfCAP-H w/ SYTOX PfCAP-H w/ NHS-Ester Merge .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 7: PfCAP-H knockout affects karyokinesis but not cytokinesis. Synchronized PfCAP-HDiCre 352 parasites were treated with rapamycin or DMSO, probed with α -V5 (PfCAP-H, magenta), α -PfGAP45 353 (IMC, green), NHS-Ester (protein, greyscale), and SYTOX (nucleus, cyan) for U-ExM (n= 3). (A and B) 354 E64-arrested late matured schizonts, PfCAP-H KO parasites showed giant agglomerate of incompletely 355 separated nuclei while the IMC appeared to be surrounding all potential merozoites, implying unaffected 356 cytokinesis. Scale bar = 2 µm. 357 358 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint in both PfCAP-H iKO and control parasites. In addition, the absolute gametocytemia on day 6 359 after induction was similar at 1.9 +/ - 0.3% and 2.0 +/ - 0.1% (mean +/ - SD) in the DMSO and 360 rapamycin conditions, respectively. In stage III gametocytes, PfCAP-H localizes adjacent to the 361 nucleus (Fig. 8A). Intriguingly, we did not find any significant morphological difference between 362 PfCAP-H iKO and control parasites throughout the 12 days of the assay (Fig. 8B, S8A, and 363 S8B). 364 365

366 To unravel the function of PfCAP -H during the erythrocytic development in Plasmodium 367 falciparum, we employed an inducible knockout parasite strain and ultrastructure expansion 368 microscopy. We revealed that PfCAP -H is essential for asexual blood stages. The PfCAP-H 369 knockout exhibited robust nuclear division defect , while the cytokinesis continued relatively 370 normally. However, this is distinct from most eukaryotes, where the surveillance system verifies 371 that nuclear division is completed typically before the onset of cytokinesis [38, 39]. Thus, this 372 work corroborates previous studies that showed that karyokinesis and cytokinesis are 373 independent, and these malarial parasites lack some aspects of the surveillance systems 374 present in other eukaryotes [29, 40, 41]. 375 376 In many eukaryotes, condensation of chromosomes during mitosis is a crucial step to ensure 377 the faithful division of the genomic material in daughter cells. However, it is intriguing to 378 comprehend how this equal distribution of genome is maintained and regulated in lower 379 eukaryotes that possess decondensed forms of chromosomes throughout their life cycle [42]. 380 Remarkably, despite the absence of condensed chromosomes, these lower eukaryotes have 381 retained the highly conserved condensin complexes, which facilitate condensation of 382 chromosomes [11]. This forces us to ponder the significance of these complexes in such an 383 unusual scenario. So far, the function of condensin complexes in Saccharomyces cerevisiae has 384 been extensively studied to decipher the equal distribution of the genome with such atypical 385 decondensed chromosomes during closed mitosis [27, 43-45]. These complexes are recently 386 identified in Plasmodium spp., which share decondensed chromosomes similar to fission yeast 387 [2, 14, 16]. However, attributing to their small nuclear size (~1 µm diameter) and underexplored 388 molecular mediators during mitosis compared to model organisms [29], it is challenging to 389 investigate the chromosome dynamics in Plasmodium spp. during mitosis. The foremost step in 390 the quest of interrogating chromosome dynamics is to get detailed evidence of different mitotic 391 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint A PfCAP-H w/ PfGAP45Merge B 0 25 50 75 8D 8R 10R 12R12D10D Percentage (%) Stage I - II Stage III Stage IV Stage V Aberrant 100 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 8: PfCAP-H is likely dispensable for gametogenesis. (A) Slide-based IFA was performed for 392 the localization of PfCAP-H in sexual stages (Stage III) on Day 8 post-induction. The slides were probed 393 with α-V5 (magenta) for PfCAP-H and α-PfGAP-45 as IMC markers (green). IFA showed that PfCAP -H 394 is present adjacent to the nucleus in stage III gametocytes. Scale bar = 2 µm. (B) The progression of 395 gametogenesis was examined in PfCAP-HDiCre parasites, treated with rapamycin or DMSO, from Day 8 396 to Day 12 post-induction, by calculating different stages (Stage I to Stage V) with Hemacolor stained thin 397 smears. The aberrant category includes parasites that appear stressed, distorted, and abnormal 398 parasites. In contrast, the unknown category comprises the gametocytes, which were difficult to 399 categorize based on standard categorization by Carter et al., 1979 . The experiment was performed in 400 two independent biological replicates. The error bar indicates SEM calculated by GraphPad Prism. 401 402 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint stages in these parasites. Strikingly, t he recent establishment of ultrastructure expansion 403 microscopy techniques in these parasites has unlocked the feasibility of studying the nuclear-404 related processes in these parasites [46]. Leveraging U -ExM capability to expand the size of 405 parasites, we captured highly resolved images of mitotic events in these parasites. Furthermore, 406 we showed that the PfCAP-H is highly expressed at the metaphase plate and propose that 407 PfCAP-H could be used as a marker for the metaphase plate to mark the mitotically active 408 nucleus during schizogony. Consequently, subcellular localization of PfCAP-H at the metaphase 409 plate, along with the cues from localization of the mitotic spindle, as in the yeast mitosis model 410 [43], would further aid in advancing our knowledge on mitosis in malarial parasites. 411 412 This study demonstrates that PfCAP-H interacts with PfCAP -G and is a crucial member of the 413 condensin I complex. The U -ExM revealed that PfCAP -G do es not localize to the mitotic 414 chromosome in the absence of PfCAP-H, suggesting that PfCAP-H is required to load PfCAP -415 G on the mitotic chromosome, similar to its homolog s in Drosophila and human s [18, 26] . 416 Furthermore, t he sequence analysis of PfCAP -H showed that PfCAP -H contains highly 417 conserved N and C terminal region, which are the sites of interaction for SMC2/4 in its homologs 418 [11, 21], inferring that PfCAP-H might also bind to SMC2/4 via these highly conserved regions. 419 In P. berghei, it was shown that PbCAP-H co-immunoprecipitates with SMC2/4 in late schizonts 420 but not in early schizonts [14]. This information hints that SMC2/4 can bind to chromosomes 421 alone; however, PfCAP-H is required for assembling the subunits to function as a condensin I 422 holo-complex. We propose that PfCAP-H plays a vital role in the assembly of the condensin I 423 complex on the mitotic chromosomes in Plasmodium parasites, comparable to its homolog in 424 other systems [17]. Furthermore, the essential ity of this complex suggest s that chromosome 425 condensation does occur in these parasites during mitosis, albeit to a lesser extent than in higher 426 eukaryotes. 427 428 PfCAP-H is dynamically localized during schizogony with peak expression during its growth 429 phase, followed by diffuse localization at the end of the schizogony. This expression and 430 localization resemble the similar pattern displayed by the condensin I complex in several other 431 eukaryotes during mitosis [9]. In model organisms, condensin complex localization and activity 432 are tightly regulated through phosphorylation by mitotic kinases [47]. For example, the mitotic 433 kinase cdc2-cyclin B phosphorylates the non-SMC subunits of condensin in Xenopus and human 434 cells to condense the chromosome [48]. Barren, the Drosophila homolog of PfCAP -H, 435 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint recruitment to mitotic chromosomes is facilitated by phosphorylation by Aurora B kinase [49]. 436 Notably, phosphoproteomic analysis in Plasmodium falciparum showed that all the subunits of 437 the condensin I complex, including PfCAP-H, are phosphorylated during asexual blood stages 438 [6, 50]. We thus speculate that the condensin I complex is likely regulated by phosphorylation in 439 these parasites. However, further study is required to investigate the mechanisms for controlling 440 condensin I activity in Plasmodium falciparum. 441 442 Interestingly, while scrutinizing the localization of PfCAP -H in blood stages, we observed that 443 some schizonts exhibited unconventional schizogony where already duplicated nuclei restart the 444 next round of mitosis before completing karyokinesis. This observation agrees with previous 445 studies of asexual blood stage development of P. falciparum [7, 51]. This variation in schizogony 446 has been observed in the mosquito midgut during sporozoite formation of Plasmodium spp. [52] 447 and is described as schizogony with limited karyokinesis [4]. Conversely, the evidence of such 448 variation in the asexual blood stage suggests that schizogony is somewhat fluid and is not 449 restricted only to classic schizogony. However, the factors responsible for such flexibility in blood 450 stages remain to be explored. 451 452 In summary, we have shown that PfCAP-H is essential for the asexual blood stage development. 453 PfCAP-H is critical for assembl ing the condensin I complex on the mitotic chromosomes. The 454 knockout of PfCAP-H results in agglomeration of nuclei, possibly due to improper chromosome 455 segregation, and defective karyokinesis – while cytokinesis remains largely normal (Fig. 9). 456 Furthermore, PfCAP-H is not required for centrosome duplication or mitotic spindle assembly 457 during schizogony. Overall, this study sheds light on the function of the condensin I complex 458 during mitosis in these parasites and emphases their importance in lower eukaryotes with 459 primitive mitotic features of decondensed chromosomes. 460 461 462 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Multiple rounds of DNA replication without karyokinesis Multiple rounds of DNA replication, then karyokinesis ΔPfCAP-H PfCAP-H PfSMC2 PfSMC4 PfCAP-G PfCAP-D2 Rhoptries PfCAP-H Nucleus Merozoite .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Figure 9: Proposed model of PfCAP -H function during blood stage development. Schematic 463 representation of the proposed mechanism by which PfCAP -H function during schizogony. PfCAP -H 464 plays a pivotal role in facilitating the formation of condensin I complex on the mitotic chromosomes. In 465 parasites lacking PfCAP -H, the process of karyokinesis is significantly impaired, while the cytokinesis 466 remains unaffected. 467 468 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint

469 470 Plasmid construction: 471 pPG25 (for smV5 tagged PfCAP -H): The 5’ homology region (HR) and 3’ HR region were 472 amplified from Pf3D7 genomic DNA with oJDD6763/6764 and oJDD6766/6767, respectively. 473 The codon-altered PfCAP-H gene block s (GB70.2 and GB71, IDT DNA) were amplified with 474 oJDD6598/6599 and oJDD6196/6197, respectively. The smV5 and hDHFR region was amplified 475 from pPG12 with oJDD6765/6086. The pGEM backbone for this plasmid was amplified from 476 pPG12 with oJDD6083/6084. The six pieces were assembled with the Golden Gate BsaI-HF v2 477 assembly kit (NEB). Three guide RNA (gRNA) plasmids were used to target the PfCAP-H locus. 478 The guides were annealed and ligated into BpiI digested pRR216 plasmid (SpCas9 expression 479 plasmid) to construct pJPM59 (oJDD4921/4922), pCJM57 (oJDD5791/oJDD5792), and pCJM58 480 (oJDD5793/oJDD5794), respectively. The EcoRI/NotI linearized pPG25 plasmid was co -481 transfected with the three guide RNA -containing plasmids into 3D7 pfs47DiCre parasites to 482 generate PfCAP-HDiCre parasites. 483 484 pRR85 (for generating BirA fused PfCAP -G): The 5’ HR and 3’ HR regions were amplified 485 from 3D7 genomic DNA with primers oJDD3881/3882 and oJDD3875/3876. Codon-altered 486 PfCAP-G was amplified from gene block GB04 with oJDD3883/3884. The U6 promoter, gRNA 487 for PfCAP-G, and U6 terminator were amplified from pBAM203 were assembled by overlapping 488 PCR extension with oJDD3877/3878 and oJDD3879/3880 . These five pieces were assembled 489 by overlapping PCR and ligated into pRR28 cut with NotI and XhoI. The SpeI linearized pRR85 490 plasmid was co -transfected with a SpCas9 expression plasmid into the 3D7 parasite strain to 491 generate PfCAP-GBirA parasites. 492 493 pCJM22 (for generating smHA-tagged PfCAP-G): The PfCAP-G region was digested from 494 pRR85 with NotI/XhoI. The smHA and BSD sequence s were obtained from pCJM01 by 495 NotI/XhoI digestion. These two digested fragments were ligated with T4 DNA ligase to get 496 pCJM22. For transfection, th e Spe1 linearized pCJM22 plasmid was transfected with the 497 SpCas9 expression plasmid in PfCAP-HDiCre strain to generate PfCAP-HDiCre/PfCAP-G parasites. 498 499 pPG89 (for generating smHA -tagged PfNDC80): The PfNDC80 region was PCR amplified 500 with primers oJDD8427/8430 and digested with NotI/XhoI. The smHA and BSD sequences were 501 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint obtained from pCJM22 by NotI/XhoI digestion. These two digested fragments were ligated with 502 T4 DNA ligase to get pPG89. For transfection, the Spe1 linearized pPG89 plasmid was 503 transfected with three guide RNA-containing plasmids in PfCAP-HDiCre strain to generate PfCAP-504 HDiCre/PfNDC80 parasites. 505 506 All primers and geneblocks are shown in Supplemental Table S2. 507 508 Reagents and Antibodies 509 All the primers used in this study were purchased from Thermo Fisher, gene blocks were 510 purchased from IDT DNA, and restriction enzymes were purchased from New England Biolabs. 511 The primary antibodies used in this study are the following: mouse a-V5 (clone SV5-Pk2, Bio-512 Rad); rabbit a-V5 (ICL, RV5-45A-Z), rat a-hemagglutinin (HA, clone 3F10, Sigma); mouse a-513 tubulin (clone B-5-1-2, Sigma); rabbit a-Histone H3 (ab1971, Abcam); mouse a-Centrin (CrCen 514 clone 20H5, EMD Millipore); and rabbit a-PfGAP45 (gift from Julian Rayner at the University of 515 Cambridge [53]). Secondary antibodies and other reagents (Alexa Fluor 405 NHS-Ester, SYTOX 516 Deep Red Nucleic acid stain, and Hoechst 33342 solution) used for microscopy were purchased 517 from Thermo Fisher. 518 519 Sequence Alignments 520 The FASTA sequence of Plasmodium falciparum PfCAP-H (PF3D7_1304000) and its orthologs 521 including Plasmodium berghei PbCAP-H (PBANKA_1402500), Saccharomyces cerevisiae 522 BRN1 (P38170), Schizosaccharomyces pombe CND2 (Q9Y7R3) Arabidopsis thaliana CAPH 523 (Q564K3), Xenopus laevis NCAP-H (O13067), Homo sapiens NCAP-H (Q15003), and 524 Drosophila melanogaster Barren (P91663) were obtained from UniProt or PlasmoDB. The 525 sequence identity and similarity was calculated using EMBOSS Needle [54]. Multiple sequence 526 alignment was carried out using the default MUSCLE algorithm with MEGA software and aligned 527 sequences were analyzed using ESPript 3.0 [55]. 528 529 Plasmodium falciparum culture and transfection 530 The Plasmodium falcip arum 3D7 strain was obtained from the Walter and Elizabeth Hall 531 Institute, and the 3D7pfs47DiCre parasite strain was obtained from Dr. Ellen Knuepfer [54]. 532 Parasites were cultured in human O+ erythrocytes (deidentified from commercial vendor) at 4% 533 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint hematocrit (HCT) in RPMI-1640 (Sigma) supplemented with 25 mM HEPES (4-(2-hydroxyethel)-534 1-piperazineethanesulfonic acid) (EMD Biosciences), 0.21% sodium bicarbonate (Sigma), 50 535 mg/l hypoxanthine (Sigma), and 0.5% Albumax II (Invitrogen) and were kept at 37 °C with a 536 mixture of gases (5% CO2, 1% O2, and 95% N2). Parasite growth was monitored by staining with 537 Hemacolor (Sigma) staining solution s and observed under the microscope. The cultures were 538 synchronized by combining Percoll and sorbitol treatments [56, 57]. 539 540 To generate transgenic parasite s, 60 μg of donor plasmid was linearized by digestion and co-541 transfected with 60 μg of SpCas9-plasmids containing gRNA into 10% ring-staged P. falciparum 542 (3D7pfs47DiCre or 3D7) parasites by electroporation. The electroporation was performed at 543 settings of 310 V, 950 μF and infinite Ω in a 0.2 cm cuvette with BioRad GenePulser . The 544 parasites were cultured and selected with appropriate drugs. The PfCAP-HDiCre and PfCAP-GBirA 545 parasites were selected with 2.5 nM WR99210 (Jacobus Pharmaceuticals), and PfCAP -546 HDiCre/CAP-G parasites was selected with 2.5 ug/ml blasticidin (Research Products 547 International). Single clones of these transfected parasites were obtained by limitation dilution. 548 The transgenic parasites were verified by PCR amplification using primers and/or whole genome 549 sequencing of harvested genomic DNA (Biobasic blood genomic DNA miniprep kit). To induce 550 excision of PfCAP-H gene, 0 – 4 h young parasites were treated with 100 nM rapamycin for up 551 to 12 hrs, followed by washing with new culture media [28]. 552 553 Gametocyte induction 554 Trophozoite- stage parasites at 3% parasitemia and 2 % HCT were cultured with 50 % 555 conditioned AlbuMax II medium. After 2 days, the culture was treated with 0.25 mg/ml heparin 556 (Alpha Aesar A16198) and 2.5 mM N-acetylglucosamine to prevent subsequent invasion of 557 asexual stage parasites. Gametocyte conversion and morphology was evaluated by blood 558 smear stained with Hemacolor staining solution under light microscopy. The experiments were 559 performed in two independent biological replicates. The gametocyte conversion was calculated 560 by the ratio of gametocytes on day 6 to ring parasitemia on day 2. IFAs was performed on day 561 8 to localize the protein in gametocyte stages. The gametocytes were categorized as described 562 [58]. 563 564 Replication assay 565 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Synchronized ring-stage parasites were seeded with an initial parasitemia at 0.25 % in 1 % HCT, 566 treated with DMSO or 20 nM rapamycin, and cultured for two consecutive cycles.100 μL culture 567 was collected from each well on three different days (0, 2, and 4) and then washed once with 568 0.5% bovine serum albumin (BSA) in 1x PBS. The samples were resuspended in 100 μL staining 569 solution containing 1:1000 SYBR Green I (Invitrogen) dilution in 0.5% BSA/PBS and incubated 570 for 20 mins at RT. The stained samples were washed with 0.5 % BSA/PBS and then 571 resuspended in 1x PBS. The number of infected red blood cells was determined by flow 572 cytometry (FACSCalibur) using the CellQuest Pro program. The data from 100,000 cells was 573 analyzed by FlowJo X and GraphPad Prism 9 software and represented as mean and SD of 574 triplicates. 575 576 Immunofluorescence assays (IFA) 577 For IFA, parasites were smeared onto slides and air-dried. The entire process of IFA was done 578 in a humid chamber. The parasites were fixed with 4% (v/v) paraformaldehyde (PFA) in 1x PBS 579 for 10 mins and rinsed quickly with 1 x PBS. The fixed parasites were permeabilized with 0.1% 580 Triton X-100 in PBS for 10 mins at room temperature (RT) and then washed three times with 1x 581 PBS for 3 mins. The parasites were blocked with a 3% (w/v) BSA in PBS for 1h at RT. The smear 582 was stained with respective p rimary antibodies (mouse a-V5 1:500, rat a-HA 1:250, mouse a-583 Centrin 1:500, rabbit a-V5 1:500) for 1h at RT or overnight at 4°C, followed by washing three 584 times with 1x PBS for 5 mins. The samples were incubated with fluorescently labeled secondary 585 antibodies (1:1000) for 30-45 mins at RT and washed thrice with 1x PBS for 5 mins to remove 586 excess unbound antibodies. The DNA content of the parasites was stained with Hoechst 33342 587 (1:5000) in 1x PBS for 20 mins at RT and then quickly rinsed with 1x PBS. The parasites were 588 mounted in Vectashield Vibrance antifade mounting media (Vector Laboratories Inc. H -1700) 589 with coverslips and stored at 4°C until imaging. The z-stacked Images were acquired on a Zeiss 590 LSM900 microscope with Airyscan 2 with 63X objective and analyzed using FIJI software. 591 592 Ultrastructure Expansion microscopy (U-ExM) 593 For expansion microscopy, we followed a four-day protocol. On day 1, synchronized parasites 594 at 4-5% parasitemia were collected and allowed to settle on a poly-D-lysine coated coverslip for 595 20 mins at 37 °C. Parasites were fixed with pre -warmed 4% (v/v) PFA for 20 mins at 37 °C, 596 washed thrice with pre -warmed 1x PBS, and crosslinked with 1.4% formaldehyde and 2% 597 acrylamide solution in 1x PBS overnight at 37 °C. On day 2, the gel polymerization was done in 598 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint a gelation chamber. The parasite -coated coverslips were placed onto the mixture of 599 TEMED/APS/monomer solution, kept on ice for 5 mins, and then incubated for 1 h at 37 °C. Post 600 incubation, the coverslips plus gel were placed in a 6 -well dish with 1ml denaturation buffer for 601 15 mins with agitation to displace gel from the coverslip. The detached gel was then incubated 602 with 1.5ml denaturation buffer in a microcentrifuge tube for 90 mins at 95 °C. After cooling off , 603 gels were transferred to a petri -dish containing 25ml ddH2O and incubated for 30 mins at RT. 604 The gels were further incubated with 25 ml ddH2O overnight at RT to perform the first round of 605 expansion. On day 3, gels were washed twice with 1x PBS for 15 mins at RT. The gels were 606 blocked with 3% BSA/PBS for 30 mins at RT, followed by staining with respective primary 607 antibodies (mouse a-V5 1:250, rat a-HA 1:100, mouse a-tubulin 1:500, mouse a-Centrin 1:250, 608 rabbit a-GAP45 1:2500) in 3% BSA/PBS overnight at 4°C. On day 4, gels were washed thrice 609 with 1x PBS with 0.5% Tween-20 for 10 mins at RT with agitation. Gels were stained with their 610 respective secondary antibodies (1:500), Alexa Fluor 405 NHS-Ester (1:250), and SYTOX Deep 611 Red dye nucleic acid stain (1:1000) in 1x PBS for 2h 30mins at RT protected from light. The 612 stained gels were washed thrice with 1x PBS/0.5% Tween -20 for 10 mins at RT and then 613 incubated with ddH2O for 30 mins. Water was replaced, and gels were incubated overnight at 614 RT for the second round of expansion. The images were captured on a Zeiss LSM900 615 microscope with Airyscan 2 and analyzed using FIJI software. 616 617 Immunoblots 618 Parasites were harvested by 0.02% saponin in PBS with protease inhibitors (PIC) (SigmaFast 619 Protease Inhibitor Cocktail) and boiled in 1X Laemmli buffer supplemented with 1 x PIC for 5 620 mins at 95 °C. The protein lysate (equivalent to 10 8 parasites per lane) were run on a 4 -20% 621 Tris-glycine-sodium dodecyl sulfate gel and transferred to a PVDF membrane. The membrane 622 was blocked with Licor Odyssey blocking buffer for 1 hr at RT. The immunoblot was probed with 623 primary antibodies ( a-V5 1:1000, a-HA 1:1000, and a-H3 1:2500), followed by incubation with 624 secondary antibodies (1:1000) diluted in the Licor Odyssey blocking buffer. The immunoblot was 625 scanned on a Licor Odyssey CLx imager system and quantified using volumetric measurement 626 of fluorescence intensity with LiCor Image Studio 4.0. 627 628 BirA Biotin Proximity-labeling 629 Synchronized 44-46 hour schizont-stage parasites (PfCAP-GBirA and 3D7) were incubated with 630 150 μM biotin for 6 hours; the Protein Kinase G inhibitor, compound 1, was added to prevent 631 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint parasite egress[59]. After this incubation, parasites were harvested with 0.05% saponin in 1x 632 PBS and protease inhibitors. Parasite pellets were resuspended in 1ml of RIPA lysis buffer with 633 PIC (50 mM Tris-HCl, Ph 7.5, 150 mM NaCl, 1% NP-40 (Tergitol), 0.5% Sodium deoxycholate, 634 0.1% Sodium dodecyl sulfate) for 1 h at RT on the rotator and sonicated with microtip sonicator 635 with 30 s at 20 amplitudes, followed by 3 mins incubation on ice. This cycle was repeated twice. 636 The lysate was spun down for 30 mins at max speed to remove hemozoin and other insoluble 637 debris. The streptavidin -coated beads were washed with RIPA/PIC and incubated with the 638 cleared lysate overnight on the rotator at 4°C. After incubation, beads were sequentially washed 639 with RIPA buffer and Wash Buffer 1 (2% SDS), Wash buffer 2 (50 mM HEPES, pH 7.5, 500 mM 640 NaCl, 1 mM EDTA, 1% Triton -X 100, 0.1% Sodium deoxycholate), Wash buffer 3 (10 mM Tris 641 HCl, pH 8.0, 250 mM Lithium Chloride, 1 mM EDTA, 0.5% NP-40, 0.5% Sodium deoxycholate), 642 and Wash buffer 4 (50mM Ammonium bicarbonate in ddH20). The sample was resuspended in 643 40 μL of wash buffer 4 and stored at -80 °C until further processing for mass spectrometry. On-644 bead digestion, followed by LC/MS-MS and data analysis, was performed at the Harvard Taplin 645 Mass Spectrometry Facility. The results were analyzed by comparing the unique and total 646 peptides between PfCAP-GBirA and 3D7 parasites in three independent biological replicates. 647 648

649 We acknowledge the support of Ross Tomaino at the Taplin Mass Spectrometry Facility and 650 Paula Montero-Llopis of the Harvard Medical School MicRoN core. This work was supported by 651 the National Institutes of Health R01 AI145941 (J.D.D). 652 653 Author Contributions 654 P.G.: Conceptualization, Methodology, Formal Analysis, Investigation, Writing –Original Draft 655 Preparation, Visualization. J. P.M.: BioID experiment and analysis. J.D.D.: Conceptualization, 656 Data Curation, Supervision, Project Administration, Funding Acquisition, Writing –Review and 657 Editing. 658 659 The authors declare no competing interests. 660 661 662 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint Data Availability 663 All data generated throughout this study are incorporated into the manuscript and supplementary 664 files. Whole genome sequencing data have been deposited in the NCBI Sequence Read Archive 665 (#XXXXX). Protocols, raw data, or any materials employed in this study are available upon 666 request. 667 668 Author ORCIDs 669 Pratima Gurung: https://orcid.org/0000-0002-5197-1881 670 Jeffrey D. Dvorin: http://orcid.org/0000-0002-5883-7271 671 672 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 29, 2024. ; https://doi.org/10.1101/2024.02.26.582160doi: bioRxiv preprint

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