Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii

Abstract

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

Introduction

Apicomplexan parasites replicate by strategies that differ in nuclear division, but are similar with respect to the formation of new daughter parasites by a unique process of internal budding. Apicomplexans are remarkably accomplished specialists of multinuclear replication. In schizogony, parasites form multiple nuclei by alternating rounds of DNA synthesis and mitosis with cytokinesis delayed until the last round of nuclear division [1]. Endopolygenic division is a variation on schizogony where multiple rounds of chromosome replication occur within a single (polyploid) nucleus that undergoes concerted nuclear division and parasite budding at the end of the cycle [2], [3]. By contrast, endodyogeny is a simple binary division with a single chromosome replication followed by concurrent mitosis and parasite budding [4]. Cytokinesis in endodyogeny occurs through a unique internal assembly of daughters that is very similar to the final cycle of schizogony in other apicomplexans suggesting that there is a common set of cell cycle controls that operate in both division strategies [5], [6].

The comparative simplicity of endodyogenic division and strengths of the Toxoplasma experimental model system have allowed the major phases of the tachyzoite cell cycle and the basic order and timing of nuclear and organelle division to be established [7]. Toxoplasma tachyzoites divide using a three-phase cycle comprised of a G1 (60%), S (30%) and mitosis (10%) with the classic G2 period between S phase and mitosis either very short or absent [8], [9]. The length of the S phase period in the tachyzoite cell cycle has been independently verified by [H³]-thymidine radiolabeling, time-lapse microscopy of transgenic parasites expressing TgPCNA1-GFP (monitoring replication foci), and by calculating the length of S phase from genomic DNA analysis of synchronized populations [8]. It is not yet possible to pinpoint the start of mitosis, although conoids appear to form at this time [8], [10] and replication foci dissolve <1 h before nuclear division consistent with a minimal or absent G2 period [8]. This finding parallels similar observations in Theileria and other protozoa [11], [12]. S phase distributions in tachyzoites are peculiar [8], [13], with late S phase parasites (∼1.8N) more numerous than parasites in early S. It is intriguing that internal daughter budding also appears to initiate in late S phase [13], and therefore, a novel apicomplexan mechanism has been proposed that safeguards the proper timing of budding with mitosis might be responsible for the 1.8N subpopulation [8]. Establishing the molecular basis for these observations is needed to understand these unusual cell cycle distributions.

Checkpoint control of apicomplexan cell division is poorly understood, although factors commonly associated with eukaryotic cell cycle checkpoints are present in apicomplexan parasites including cyclins, CDKs, MAPKs, and components of the anaphase promoting complex [14] that regulates mitosis. While these findings predict that cell cycle controls exist in these parasites, they do not provide information about where checkpoints function or how these controls operate to coordinate the diverse strategies utilized by these parasites to proliferate. Experimental proof for active checkpoint control in Toxoplasma has been controversial [15], [16], however, there is now growing evidence for a model that proposes specific transitions in the tachyzoite cell cycle where parasites naturally halt during progression through the G1, S and mitotic phases [8], [9], [17], [18]. In this paper, we describe a new Toxoplasma mitotic mutant and identify the essential defect responsible for conditional growth through genetic complementation. The fundamental nuclear factor revealed by these studies is required for chromosome segregation in Toxoplasma and other eukaryotes establishing an ancient cell cycle mechanism conserved over hundreds of million years of eukaryotic cell evolution. The primary defect in this mutant also provides insight into the mechanistic restrictions that coordinate mitosis and the internal assembly of daughter parasites in the Apicomplexa.