EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment inPlasmodiummale gametogenesis

Abstract

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter male gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium evolves the endomitosis for this multinucleated cell division. However, the mechanism underlying the spindle-kinetochore attachment remains elusive. End-binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here we report that Plasmodium EB1 is a unique orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays revealed that Plasmodium EB1 lost MT plus-end tracking but gained MT-lattice affinity. This MT-binding feature of EB1 is contributed by both the CH domain and the linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission of parasite. EB1 is localized at the nucleoplasm of male gametocytes. Upon gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout three rounds of endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment was observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice affinity has evolved to fulfill the spindle-kinetochore lateral attachment in male gametogenesis.

A non-canonical Arp2/3 complex is essential forPlasmodiumDNA segregation and transmission of malaria

“…Notably, only about 30-60% of all microgametes contained any detectable DNA signal, supporting previous notions that DNA segregation into male gametes is an error-prone process ( Fig. 2G ) ( 32 , 33 ). Activated male gametocytes of both Pb 820 WT and Pb 820 ARPC1(-) lines had the expected octoploid DNA content ( Fig.…”

“…A core protein connecting the kinetochores to the spindle during male gametogenesis is end-binding protein 1 (EB1) ( 32 , 47 , 48 ). Deletion of EB1 in P. berghei resembles the phenotype of ARPC1(-), with an arrest in a delayed-death like manner in early oocysts ( 48 ).…”

“…Deletion of EB1 in P. berghei resembles the phenotype of ARPC1(-), with an arrest in a delayed-death like manner in early oocysts ( 48 ). In contrast, deletion of EB1 in P. falciparum and P. yoelli leads to the formation of gametes that were entirely deficient of DNA and incapable of fertilising females, highlighting interesting species-specific differences in DNA segregation in male gametocytes ( 32 , 47 ). A paternal phenotype followed by an early oocyst arrest has also been described for other Plasmodium parasite lines lacking proteins such as the formin-like protein MISFIT( 49 ), the microtubule motor kinesin-8X ( 50 ), the aurora kinase Ark2 ( 48 ), and for a parasite line that expresses actin 1 in place of actin 2 ( 44 ).…”

Expansion microscopy of apicomplexan parasites

“…The use of NHS ester, therefore, allows the visualization of a number of protein‐dense structures within the parasite (similar to electron microscopy), without the need for protein‐specific antibodies. Across various stages of the Plasmodium lifecycle, NHS ester has been used to visualize the rhoptries, some cytoskeletal elements, basal complex, cytostomes, apical polar ring (APR) and nuclear microtubule organizing centre (MTOC) (Bertiaux et al, 2021; Liffner & Absalon, 2021; Liffner et al, 2023; Rashpa & Brochet, 2022; Rashpa et al, 2023; Simon et al, 2021; Yang, Cai, et al, 2023); highlighting it is a powerful tool for generally observing changes within the parasite. Following the publication of the first study using U‐ExM in Plasmodium , further significant developments were made when the protocol was optimized for the most common Plasmodium fixative, paraformaldehyde (Simon et al, 2021), followed by the first U‐ExM experiments using P. falciparum (Simon et al, 2021).…”

“…The ability to infer the position of individual chromosomes and kinetochores will have a significant impact on our understanding of the dynamics of mitosis and meiosis in malaria parasites in future. Further, these observations provide a platform for the characterization of proteins involved in mitosis and meiosis, with a recent study using U‐ExM to show that end‐binding protein 1 (EB1) mediates microtubule–kinetochore attachment during mitosis of gametocytes (Yang, Cai, et al, 2023).…”