Mutant/mutation
The mutant lacks expression of ARPC1. In addition, it expresses GFP under control of a male gametocyte specific promoter and RFP under the control of a female gametocyte specific promoter.

See also RMgm-5441 for a mutant lacking expression of ARPC1 and expressing RFP under the constitutive hsp70 promoter (line Pb473ARPC1(-) )

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.10.25.563799

Protein (function)
ARPC1 is a 41 kDAprotein predicted to fold into a WD40 repeat domain with two alphahelical loops extending from the doughnut-shaped β-propeller.
From the paper: 'The actin-related protein 2/3 (Arp2/3) complex is a seven-subunit complex consisting of Arp2, Arp3 and five supporting subunits ARPC1-5 that together nucleate actin filaments. In the cytoplasm of most metazoa, the Arp2/3 complex mediates lamellipodia formation and endocytic trafficking, among other processes. Nuclear functions of Arp2/3 are less well studied and differ between species, but they include DNA damage repair, nucleation of spindle actin during mitosis and meiosis and chromosome capture and segregation. Despite the evolutionary conservation of Arp2/3 across the eukaryotic kingdom, it was assumed that the complex has been lost in apicomplexan species, except for a single subunit, annotated as ARPC1/ARC40 in Plasmodium. Here we discovered that Plasmodium ARPC1 constitutes part of a highly divergent, non-canonical Arp2/3 complex, which associates with mitotic spindles in activated male gametocytes and is essential for genome segregation into budding gametes. Disruption of the complex results in a delayed death phenotype in oocysts leading to a complete transmission block within mosquitoes'.

Phenotype
Normal production of male and female gametocytes. See also below for more detailed information on the phenotype of male gametes.
Normal production of mature ookinetes with wild type morphology and motility. The DNA content of ookinete nuclei was reduced by about 30% in Pb473ARPC1(-) compared to Pb473WT.
Ookinetes were fully infective, as infecting mosquitoes with Pb473ARPC1(-) led to WT-like infection rates (prevalence) and oocyst numbers at early development (6 days post infection (dpi). However, oocyst numbers dropped significantly during later oocyst development (12 dpi). None of the oocysts sporulated, and we detected no sporozoites in midguts or salivary glands. No infection of mice after bites of infected mosquitoes.
We imaged WT and KO oocysts at 4, 6, and 12 dpi and found Pb473ARPC1(-) oocysts to be significantly smaller compared to WT at all time points. Pb473ARPC1(-) oocysts also exhibited a lower DNA content than WT oocysts.

Additional information
Analysis of a mutant expressing a C-terminal GFP-tagged version of ARPC1 (RMgm-5442) showed mainly expression in gametocytes and ookinetes. In both stages, PbARPC1 localises to the nucleus. A weak ARPC1-GFP signal was also observed in late stage oocysts.
To test whether ARPC1 expression is restricted to males, we tagged ARPC1 with an HA-tag RMgm-(5443) in the gametocyte reporter line Pb820WT. Both RFP-positive female and GFP-positive male gametocytes expressed ARPC1-HA in the nucleus, confirming the original observation with the PbARPC1-GFP line. However, we detected both by IFA and by flow cytometry a stronger ARPC1-HA signal in males compared to females, indicating a higher protein expression in the male lineage, in line with the male phenotype of PbARPC1(-).

To investigate If ARPC1 function is sex-specific, we crossed Pb(WT473)ARPC1(-) parasites with either Pb47(-) parasites that do not produce fertile females or with Pb48/45(-) parasites that do not produce fertile males. Only crossing with female-deficient (male-competent) Pb47(-) restored oocyst size at 6 dpi, while oocysts of the cross with male-deficient (female-competent) Pb48/45(-) parasites remained small. ARPC1 is thus required for male, but not female fertility.

Evidence is presented for:
- ARPC1 is required for correct chromosome condensation and segregation into male gametes. Our data suggests that ARPC1 is not required for spindle or gamete formation but involved in the process of DNA condensation right before budding of the flagellated gamete. Deletion of ARPC1  results in sub-haploid microgametes that contribute less than one complete genome to the zygote, suggesting that ARPC1 is responsible for proper DNA segregation into the developing male gametes.
- While ARPC1 is essential for DNA segregation during male gametogenesis, subsequent sub-haploid gametes are still fertile and ARPC1(-) parasites are able to undergo meiosis and ookinete formation.
- ARPC1 is part of a functional non-canonical Arp2/3 complex
- To further investigate the function of PbARPC1 and identify putative interaction partners, we performed immunoprecipitation of ARPC1-GFP from purified non-activated and activated gametocytes. PbGFPcon gametocytes that constitutively express nucleocytosolic GFP served as control. While in non-activated PbARPC1-GFP gametocytes, only ARPC1 was identified to be significantly enriched, we found five additional proteins to be enriched in PbARPC1-GFP activated gametocytes compared to PbGFPcon. These proteins included two actin-like proteins, annotated as Alp5a (PBANKA_0811800) and Alp5b (PBANKA_1007500), two proteins of unknown function (PBANKA_1014200 and PBANKA_1229300), and the apicomplexan-specific kinetochore protein 7 (AKiT7, PBANKA_0612300). All the identified proteins are conserved across Plasmodium and are specifically expressed in male gametocytes according to the malaria cell atlas, a single cell RNA-seq resource. Structure prediction of all identified proteins using Alphafold suggests that Alp5a and Alp5b are structurally similar to human Arp2 and Arp3, respectively.
- To test if the identified ARPC1 interaction partners form a complex and function in a similar  manner as PbARPC1, we first tagged PbARPC2 (PBANKA_1014200)(RMgm-5444) internally with YFP. Imaging PbARPC2-YFPint revealed a gametocyte-specific expression and a  localisation mirroring that of ARPC1: After activation, PbARPC2 relocalises from the nucleoplasm to the spindle, to ultimately retreat to the residual body upon DNA condensation. We also generated a knockout line of Alp5b (PbArp3) in Pb473WT (RMgm-5445). As for ARPC1(-) parasites, this parasite line showed no decrease of blood stage growth or gametocyte production but imaging of activated Pb473Alp5b(-) gametocytes and gametes revealed a significant decrease in the DNA content of male gametes.
- To visualize actin filaments, we expressed an actin-targeting chromobody fused to GFP-emerald together with mScarlet-tagged ARPC1 in P. berghei (RMgm-5446). Live imaging showed dynamic co-localisation of ARPC1 and F-Actin throughout the entire three rounds of  endomitosis. Taken together, the co-localisation of ARPC1 with F-actin and the sensitivity of male DNA segregation to cytochalasin D suggest that the divergent Plasmodium Arp2/3 complex indeed nucleates nuclear F-actin to facilitate genome segregation.

Other mutants