Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal 3xHA/GFP-tagged version of NUP221
Protein (function)
Unlike closed mitosis in yeast, P. berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm which results in a multinucleated organism prior to formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organiza on. Here we ulize ultrastructure expansion microscopy (U-ExM) to investigate tagged P. berghei nucleoporins NUP138 (PBANKA_0417900), NUP313 (PBANKA_1310200) and NUP221 (PBANKA_0416300) at the single nucleus level throughout the 24-hour blood-stage replication cycle. To visualize nucleoporins (Nups) at single NPC resolution in P. berghei, we introduced C-terminal endogenous epitope tags on three previously identified P. berghei FG Nups: spagheti monster Hemagglutinin (smHA) tags on Nup138 and Nup313 and a 3xHA/GFP tag on Nup221.
Our findings reveal that these Nups are evenly distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, which is responsible for intranuclear microtubule nucleation during mitosis.
By adapting the recombinaton-induced tag exchange (RITE) system to P. berghei, we provide evidence of NPC maintenance, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replica on of P. berghei parasites
Phenotype
In this study, we employed U-ExM to visualize individual NPCs using tagged versions of P. berghei Nup138, Nup221, and Nup313. We successfully localized these Nups to the nuclear envelope by employing NHS ester or BodipyFL Ceramide staining throughout the asexual life cycle of the P. berghei parasite. Consistent with prior findings using “serial surface” imaging in focused ion beam – scanning electron microscopy (FIB-SEM), we observed an increase in NPC number until the parasite's first mitosis, during which the NPCs were then distributed between the newly developing nuclei. We proceeded to assess NPC assembly and protein turnover using the Recombination Induced Tag Exchange (RITE) system (using NUP221) marking the first time this has been accomplished in Plasmodium. The RITE system uses a conditional, hormone activated Cre-recombinase to generate permanent epitope tag switch in the target coding sequence NUP221. This method has been previously utilized in human cells to observe the dynamic replacement of NPC components in human neurons.
Leveraging the enhanced resolution of U-ExM coupled with the RITE system, we were able to monitor NPC assembly and maintenance dynamics within a single nucleus during Plasmodium replication. In line with previous observations in mammalian cells, we found that NPCs in P. berghei undergo protein maintenance after the NPCs have been produced. Remarkably, we also confirmed the presence of Nup221 proteins of different ages in a single NPC by colocalization of original and induced Nup221 signal.
Additional information
In the early stages of the parasite's life cycle, before the parasites begin their mitotic phase (1n, Ring), we observed Nup138 being uniformly distributed around the nucleus, while the Nup313 signal is concentrated at either end of the nucleus . At this stage, we noticed a higher number of Nup313 foci (approximately 40-50 NPCs) compared to Nup138 (around 25-35 NPCs) (Fig. 3b). As the parasites began mitosis (1n, Trophozoite premitotic and mitotic), there was a significant 5-fold increase in both Nup138 and Nup313 numbers. The parasites produce over 250 NPCs containing Nup313 and over 150 NPCs containing Nup138, distributed around the single nucleus. As more nuclei are formed (2-4n Schizont, 5-9n Schizont, 10+n Cytokinesis), the number of NPCs around each nucleus decreased. Initially, approximately 250 NPCs surrounded a single nucleus, which then reduced to around 90-100 NPCs around each of the 2 or 3 nuclei. Eventually, the count further decreases to approximately 25-30 NPCs around 10-12 nuclei. Our data suggests that NPC production peaks before mitosis and NPCs are then distributed during each karyokinesis.
Quantification of NPCs around the centriolar plaque reveals a higher count of Nup313 compared to the other two FG Nups
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