A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division
Figure 9
Disruption of ISP2 results in a severe loss of parasite fitness and defects in daughter cell formation.
A. Western blot analysis using polyclonal anti-ISP2 confirms the loss of ISP2 in Δisp2 parasites. ROP1 serves as a loading control. B. A competition growth assay reveals a severe fitness loss in Δisp2 parasites. Parent and Δisp2 parasites were mixed in culture and passaged. At each passage, the composition of the mixed culture was evaluated by IFA. Although Δisp2 parasites initially comprised >80% of the culture, they were rapidly out competed by the parental strain and essentially lost from the culture within four passages. Values represent means ± 3 standard deviations. C. Parasites lacking ISP2 assemble >2 daughters per round of endodyogeny. ISP1 was used as a marker for daughter buds. The top left parasite in this vacuole is assembling four daughters while the other three parasites are assembling five daughters each. While the top left parasite has divided its nucleus and is now budding two daughters around each of two separate nuclei, the other three parasites appear to each be budding five daughters around a single polyploid nucleus. Red: mAb 7E8 detected by Alexa594-anti-mouse IgG. Green: anti-tubulin antibody detected by Alexa488-anti-rabbit IgG. Blue: Hoechst stain. D. Quantification of the >2 daughters phenotype in Δisp2 parasites. Parasites undergoing endodyogeny were counted and scored for the percentage of vacuoles in which parasites were assembling >2 daughters. Most vacuoles contain one or more parasites assembling >2 daughters in the Δisp2 strain. Values represent means ± SD, n = 3, from a representative experiment. *P<0.001. E–F. Parasites lacking ISP2 can perform karyokinesis before budding. (E) ISP1 is an early marker for bud formation visible before nuclear segregation during endodyogeny. No daughter ISP1 signal is visible in these parasites although the spindle apparatus is assembled and has already separated the chromosomes into two nuclei, showing that karyokinesis can precede budding in Δisp2 parasites. (F) After undergoing a round of karyokinesis without budding, Δisp2 parasites can bud around each of the segregated nuclei. This vacuole contains two parasites (dashed outlines) that have undergone karyokinesis prior to budding and are now assembling two daughters around each individual nucleus. Red: mAb 7E8 detected by Alexa594-anti-mouse IgG. Green: anti-tubulin antibody detected by Alexa488-anti-rabbit IgG. Blue: Hoechst stain. G–H. Parasites lacking ISP2 display catastrophic replication defects. Parasites were stained for the apicoplast thioredoxin-like protein 1 (ATrx1), which labels the apicoplast, as well as tubulin and DNA. (G) In the vacuole shown, a few parasites have received both a nucleus and an apicoplast (arrow) while others contain only an apicoplast (double arrowhead) and some contain neither (arrowhead). Several nuclei have been extruded into the vacuole along with one or more apicoplasts. (H) Other vacuoles containing extruded nuclei also contained several daughter buds that appear to be outside of an intact mother parasites (∼18 in this vacuole visible by tubulin). Many nuclei and elongated apicoplasts are present in the vacuole and appear associated with the forming buds. For clarity, a dashed line indicates the boundary of the parasitophorus vacuole. Red: anti-ATrx1 detected by Alexa594-anti-mouse IgG. Green: anti-tubulin antibody detected by Alexa488-anti-rabbit IgG. Blue: Hoechst stain. All scale bars = 5 µm.