In this study, we present the discovery of an atypical Plasmodium Arp2/3 complex required for maintaining kinetochore–spindle attachment during male gametogenesis and consequently correct DNA segregation into emerging gametes (Fig. 6). The complex localizes to the spindles during male endomitosis and remains in the residual body upon gamete budding. In the absence of individual Arp2/3 complex subunits, male gametes are subhaploid. Although they can still fertilize females to form zygotes and motile ookinetes, these mutant parasites arrest at the oocyst stage, leading to a complete block in transmission.
Fig. 6: Proposed model of the Plasmodium Arp2/3 complex during DNA replication in male gametes.
The Arp2/3 complex localizes to the spindles during the three rounds of endomitosis and then remains in the residual body upon gamete budding. The complex interacts with AKiT7 and localizes near the spindle, possibly nucleating actin 2 filaments that localize along the spindle. During the three rapid rounds of mitosis, this spindle actin may stabilize the kinetochore–spindle attachment, resulting in the formation of haploid gametes. In the absence of Arp2/3 or after inhibition of actin polymerization, no spindle actin is nucleated. The forces mediated by the repeated shortening of the spindle possibly lead to a loss of kinetochore–spindle attachment for a subset of chromosomes, resulting in the formation of subhaploid gametes. Question marks indicate open questions regarding the precise link between Arp2/3 and the kinetochore and whether Arp2/3 indeed nucleates spindle actin. EPM, erythrocyte plasma membrane; MTOC, microtubule-organizing centre; PVM, parasitophorous vacuolar membrane; PPM, parasite plasma membrane.
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Arp2/3 subunits co-localize with F-actin, and inhibition of actin polymerization during gamete formation phenocopies their deficiency. These findings are consistent with a model in which Plasmodium Arp2/3 functions as an actin nucleator, but direct biochemical evidence is still required to support this hypothesis. It also remains to be investigated whether the observed actin filaments are linear or branched. Plasmodium encodes for two actin isoforms—ubiquitously expressed actin 1, and actin 2, which is expressed in mosquito stages only. Actin 2 is essential for male gametogenesis, localizes both to the cytoplasm and the nuclear spindle of the male gametocyte and in its absence, gametocytes do not exflagellate43,49. Although complementation of actin 2 with actin 1 restores gamete formation and exflagellation, parasites still arrest in early oocysts, suggesting that actin 1 complements cytoplasmic, but not nuclear functions of actin 2 (ref. 50). We thus hypothesize that Arp2/3 facilitates polymerization of actin 2 along the spindle during male gametogenesis. Similar to Arp2/3, actin 2 is only found in the genus Plasmodium, suggesting a specialized function of this actin isoform together with the Plasmodium Arp2/3 complex.
In the absence of Alp5b (Plasmodium Arp2), ARPC1 or ARPC2, parasites form male gametes containing only half of the genome. Our data suggest that this phenotype is caused by the partial detachment of NDC80-positive kinetochores from the spindles during the second and third round of mitosis, while the initial attachment of kinetochores to the spindle is not affected. During Plasmodium male gametogenesis, kinetochores remain attached to the spindle throughout three consecutive, rapid rounds of mitosis34,48. Arp2/3-mediated actin polymerization might be required to stabilize the attachment of kinetochores to the spindle against the mechanical forces occurring during mitosis (Fig. 6). In the absence of Arp2/3, the pulling forces from the retracting spindles may cause the dissociation of individual kinetochores, leading to the retention of a subset of chromosomes in the residual body upon gamete emergence (Fig. 6). The localization of Arp2/3 subunits and F-actin to the mitotic spindle and the disrupted kinetochore localization in exflagellating ARPC1(−) or CytoD-treated parasites suggest that Arp2/3 and F-actin act near the kinetochore–spindle interphase. Yet, we did not find evidence for a direct protein–protein interaction with core kinetochore components.
Recent studies have implicated the Arp2/3 complex in the mitosis of metazoan cells, where Arp2/3 localizes to the centrosome to nucleate actin filaments permeating the mitotic spindle20,21,22,25. Inhibition of Arp2/3 perturbed centrosomal microtubule organization and impaired mitotic spindle formation and chromosome congression, leading to chromosomal segregation defects20,21,23,25. Spindle actin is also required for correct chromosome segregation during meiosis51. In contrast to metazoan Arp2/3, Plasmodium Arp2/3 localizes directly near the spindle itself. Even though the specific mechanisms vary, a role for Arp2/3 and F-actin in chromosome segregation during mitosis thus appears to be a common theme across the eukaryotic kingdom. Of note, in animal cells, the Arp2/3 complex is also implicated in DNA damage repair and safeguarding replication forks during replicative stress18,19,52. While our current data favour a role in kinetochore–spindle attachment, it cannot be excluded that Plasmodium Arp2/3 is also important to safeguard DNA integrity during the three rapid rounds of DNA replication.
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A core protein connecting the kinetochores to the spindle during Plasmodium male gametogenesis is end-binding protein 1 (EB1)34,53,54. Deletion of EB1 in P. berghei resembles the phenotype of ARPC1(−), with an arrest in early oocysts54. Interestingly, deletion of EB1 in P. falciparum and P. yoelli leads to the formation of gametes that lack DNA entirely and are incapable of fertilizing females, highlighting species-specific differences34,53. A paternal phenotype followed by an early oocyst arrest has also been described for Plasmodium parasites deficient in MISFIT, the microtubule motor kinesin-8X (ref. 55), the aurora kinase Ark2 (ref. 54), the SUN domain protein SUN1 (ref. 33), and for a parasite line that expresses actin 1 in place of actin 2 (ref. 50). Notably, MISFIT is a formin-like protein56, and in other systems, Arp2/3 and formins are known to work in concert to regulate the formation of actin networks57,58. Also, immunoprecipitation of Ark2 and EB1 in P. berghei revealed many interacting proteins including AKiT7 but not a single subunit of the Arp2/3 complex54, suggesting additional AKiT7 functions and interactions including as a linker of the Arp2/3 complex to other complexes.
Although for the above-mentioned proteins a mechanistic link with the Arp2/3 complex or Arp2/3-mediated actin polymerization remains to be determined, a unifying feature of these paternal defects is that they do not affect parasite development until the oocyst stage. This delayed-death-like developmental arrest remains puzzling. Arp2/3-deficient oocysts are equipped with a complete genome provided by the female gamete, regardless of the nature of the missing male DNA. It is possible that the aneuploidy of the oocyst leads to activation of a post-meiotic replication checkpoint that causes a cell cycle arrest. Alternatively, absence of the male genome could cause gene dosage effects that affect oocyst development. A set of paternally imprinted genes could also be expressed from the paternal genome only, as described in some plants where paternal expression of specific genes is essential after fertilization59.
Phylogenomic analyses across apicomplexan parasites for actin-related proteins and the apparent lack of branched actin in Plasmodium previously led to the prevailing assumption that the Arp2/3 complex had been lost in this phylum8,9. While the canonical Arp2/3 complex consists of seven subunits, we have so far only identified five subunits. We did not find evidence for putative Plasmodium ARPC3 and ARPC5 subunit orthologues in any of our three pulldowns or by structure-based search. Their existence cannot be excluded, as they may exhibit weaker interactions with the complex and thus be undetectable in our experimental conditions and/or be too divergent for identification by structure-based search. Instead of ARPC3 and ARPC5, we consistently identified AKiT7 as interaction partner, suggesting the formation of a hybrid complex.
Canonical Arp2/3 complexes are recruited and activated by nucleation-promoting factors (NPFs) that either belong to the WASP or the WISH/DIP1/SPIN90 family17. However, we could not identify any orthologues to these proteins in the Plasmodium genomes60. The mode of Plasmodium Arp2/3 activation thus remains to be determined, although it is tempting to speculate that AKiT7 could act as an activator. Intriguingly, Plasmodium ARPC4 contains a long, N-terminal domain that does not align to any Arp2/3 subunit. This extension may compensate for the possibly missing Arp2/3 subunits ARPC3 or ARPC5, or it may directly connect the complex to the spindle and compensate for the apparent lack of NPFs. The discovery of Plasmodium Arp2/3 raises the intriguing possibility that additional aberrant Arp2/3 complexes may be present in other apicomplexan lineages. Indeed, an ARPC1 orthologue has been annotated in Cryptosporidium8, and we found ARPC2 orthologues by structure search61 in Toxoplasma, Eimeria and Neospora. It is therefore likely that the Arp2/3 complex has assumed specialized functions in Apicomplexa that resulted in major sequence divergence while structural features remained conserved.
In conclusion, we present the discovery and characterization of a divergent Plasmodium Arp2/3 complex that is essential for malaria parasite transmission to the mosquito. The study sheds light on the unconventional cell division in the male gamete of a major eukaryotic pathogen. While further work is required to elucidate the precise mode of action and evolution of this atypical Arp2/3 complex, interfering with its function could provide a building block to break the vicious cycle of Plasmodium transmission.
Hentzschel, F., Jewanski, D., Sokolowski, Y. et al. An atypical Arp2/3 complex is required for Plasmodium DNA segregation and malaria transmission.
Nat Microbiol (2025).
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Hentzschel, F., Jewanski, D., Sokolowski, Y. et al. An atypical Arp2/3 complex is required for Plasmodium DNA segregation and malaria transmission.
Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-02023-6
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Tags: actin polymerization in gametesAtypical Arp2/3 complex in PlasmodiumDNA segregation in malariaendomitosis in Plasmodium life cycleimpact of Arp2/3 on malaria transmissionkinetochore-spindle attachment mechanismsmale gametogenesis in Plasmodiumrole of Arp2/3 in gamete formationsubhaploid male gametes in Plasmodiumzygote formation in malaria parasites
