Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin

Abstract

We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of alpha-tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum alpha-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

“…The biological significance of this phosphorylation is unclear as CDK phosphorylation has been shown to be involved in both activation and deactivation of the DNA replication complexes [50]. The malarial cell cycle operates under the confines of an endoreduplication process known as endomitosis wherein S and M phases alternate without intervening cytokinesis [51,52]. Interestingly, even during endoreduplication (which occurs during differentiation of mammalian trophoblasts and megakaryoblasts), CDKs are involved in regulating the endocyclic process.…”

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

“…Several papers report a wide range of different N per schizont, but some of these merely repeat previous literature statements without significant additional data (eg. N= 6-32 [45], N= 16-32 [46,47], N= 8-32 [48,49], N= 8-24 [50,51], N= 3-24 [52]. With regard to these previous data, the methods used most often have been light microscopy on Giemsa labeled smears of dehydrated, fixed and flattened cells.…”

The Apicoplast: A Review of the Derived Plastid of Apicomplexan Parasites

“…This number must be tied to the final number of nuclei produced. DNA replication and nuclear division in P. falciparum is an apparently disordered affair where synchrony of mitosis is quickly lost after the first couple of mitotic events (Read et al, 1993). This more flexible regime is probably the key to the variability in daughter cell number, and may allow schizogony to be better tuned to cell resources and fitness.…”

“…This more flexible regime is probably the key to the variability in daughter cell number, and may allow schizogony to be better tuned to cell resources and fitness. A disorderly conclusion to nuclear replication (Read et al, 1993) (rather than a co-ordinated final round of mitosis (Bannister et al, 2000)) determines the final number of nuclei, which may be an odd number. For the apicoplast to segregate such that each daughter cell receives one apicoplast, apicoplast division needs to receive some cue as to the number of nuclei and divide accordingly.…”