RNG1 is a late marker of the apical polar ring in Toxoplasma gondii

Abstract

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth.

SPM1 Stabilizes Subpellicular Microtubules in Toxoplasma gondii

“…A 1.0-kb fragment terminating before the stop codon of the SPM2 open reading frame was amplified from the Toxoplasma genome (RH strain) with primers SPM2 LIC 5= and SPM2 LIC 3= as listed in Table S1. The amplified genes were integrated into the pYFP.LIC.DHFR or pmCherry.LIC.DHFR vectors and transfected into ku80-null Toxoplasma parasites as previously described (77). Stable lines were isolated by selection in 1 M pyrimethamine and single cell cloned.…”

“…During cell division, which occurs by a closed mitosis, the poles of intranuclear spindles are associated with a specialized region of the nuclear envelope termed the centrocone or spindle pole plaque, which is labeled by MORN1 (24,30,51,75). The apical polar ring (APR), a circular MTOC found only in apicomplexan parasites, is located at the parasite apex and nucleates the subpellicular microtubules, which extend toward the parasite posterior in close association with the cytosolic face of the parasite pellicle (64,72,77). The coccidian subset of apicomplexan parasites (including Toxoplasma) builds an additional cytoskeletal element, termed the conoid, at the apex of the parasite (64,68).…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…Although subpellicular microtubules emanate from the APR, it does not contain ␥-tubulin, which is a marker of diverse MTOCs. However, two proteins, RNG1 and RNG2, have recently been characterized as APR constituents (89,108). RNG1 is a small, low-complexity, detergent-insoluble protein (108).…”

“…However, two proteins, RNG1 and RNG2, have recently been characterized as APR constituents (89,108). RNG1 is a small, low-complexity, detergent-insoluble protein (108). It is a late component of the APR, appearing only as daughters emerge, similar to glideosomeassociated proteins, which associate only with the mature pellicle (109-111).…”

“…IMCdelineated daughters emerge from the mother by adopting her plasma membrane (9). Some cytoskeletal components, including the APR marker RNG1, only associate with mature pellicle structures consisting of IMC in close association with the plasma membrane (108)(109)(110)(111).…”