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.

Cytoskeleton Assembly in Toxoplasma gondii Cell Division

“…Although the function of these myosins and actin-like proteins has not been determined, it is likely that some of these function in cell division. In support of this hypothesis is the recent description of actin-related protein 4a (ARP4a) with a role in chromosome segregation (Suvorova et al , 2012). …”

Discovery of a Splicing Regulator Required for Cell Cycle Progression

“…Temperature sensitive clone 12-109C6 was obtained by chemical mutagenesis of the RHΔ hxgprt strain [8]. Growth measurements were obtained using parasites pre-synchronized by limited invasion as previously described [10], [43]. Vacuoles in the infected plates were evaluated over various time periods with average vacuole sizes determined at each time point from 50–100 randomly selected vacuoles.…”

“…The following primary antibodies were used at the indicated dilutions: mouse monoclonal anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA), anti-centrin 26-14.1 (kindly provided by Dr. Jeffrey Salisbury, Mayo Clinic, Rochester, NY) and anti-IMC1 (kindly provided by Dr. Gary Ward, University of Vermont, VT) at 1∶1000. Serum raised against the conserved human centrin 1 (26-14.1) was previously shown to cross-react with the Toxoplasma centrin ortholog [10], [44]. All Alexa-conjugated secondary antibodies (Molecular Probes, Life Technologies) were used at dilution 1∶1000.…”

“…Recent reflections on the cell cycles of fungi and animals [9], indicates we should expect regulatory factor divergence even where protozoan cell cycles appear to have preserved the same network topology working to control multicellular eukaryote division. Not surprising, all expected levels of protein conservation from pan-eukaryote to species-specific growth factors are emerging from unbiased genetic screens now successfully developed for the study of Toxoplasma gondii cell division (Suvorova and White, unpublished) [8], [10], [11].…”

Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility

“…Overexpression of TgALP1 interrupted the formation of the daughter cell inner membrane complex (IMC), leading to delayed intracellular growth (17). Very recently, a new actin-related protein (ARP4) was assigned as a key nuclear protein involved in chromosome segregation in T. gondii (45).…”