Dataset Information

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.

SUBMITTER: Suvorova ES

PROVIDER: S-EPMC3767130 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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

Suvorova Elena S ES Lehmann Margaret M MM Kratzer Stella S White Michael W MW

Molecular and biochemical parasitology 20110922 1

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-le ...[more]

PMID: 21963440

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Project description:The chromatin of individual chromosomes is organized into chromosome territories (CTs) in the interphase nucleus. The spatial arrangement of CTs is non-random and evolutionarily conserved. The gene-dense and gene-poor CTs are positioned in the nuclear center and periphery, respectively. As candidates for key molecules involved in nuclear organization, we have investigated the nuclear actin-related proteins (Arps), which include the evolutionarily conserved actin-family together with conventional actin. We used a conditional knockout system with chicken DT40 cells to analyze the functions of the actin-related protein Arp6. Consistent with a previous identification of Arp6 in the SRCAP chromatin remodeling complex, the histone variant H2AZ was significantly decreased in the chromatin of Arp6-deficient cells. Most importantly, Arp6-deficient cells had impaired radial positioning of both gene-poor macrochromosome and gene-rich microchromosome CTs. A transcription microarray analysis of the cells suggests that the radial positioning of CTs impacts only a limited number of genes and plays an active role in repression, rather than in induction. As far as we know, this report is the first observation that an inner nuclear protein is required for the radial distribution of CTs, and will provide new insight into the mechanisms and physical significance of the positioning of CTs in the nucleus. The total RNA was extracted with RNeasy Maxi Kit (QIAGEN) according to the manufacturer protocol. The poly-A mRNA was isolated from total RNA using Oligotex-dT30 Super (JSR corporation) and biotinylated cRNA was synthesized according to Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix). Shortly, 2 ug of poly-A mRNA was reverse transcribed to cDNA using 100 pmol of a T7-Oligo (dT) Primer (Invitrogen), and biotinylated cRNA was prepared by in vitro transcription amplification. For hybridization, 15 ug of biotinylated cRNA was fragmented and added into a hybridization cocktail containing four biotinylated hybridization controls (bioB, bioC, bioD, and cre) as recommended by the manufacturer. GeneChip Chicken genome Arrays (Affymetrix) were hybridized with the cocktail at 45ËC for 16 hours. After washing with Non-Stringent wash buffer (6xSSPE, 0.01% Tween20) and staining with SAPE in the Affymetrix GeneChip Fluidics Station 400, the GeneChips were read using the Affymetrix GeneChip scanner 3000 7G. These systems and data analyses were operated with the GeneChip Operating Software 1.3.

Dataset Information

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

Publications

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

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