Forward Genetic Analysis of the Apicomplexan Cell Division Cycle in Toxoplasma gondii
Figure 6
Mutant V-A15 Shows Temperature Sensitive Growth due to Severe Defects in Mitosis at the Restrictive Temperature
(A) Immunofluorescence analysis of V-A15 using an anti-serum against the inner membrane complex marker IMC3 [25]: at the permissive temperature internal daughter buds (IMC3) are frequently observed, but fail to form at the restrictive temperature (36 h, quantification shown in [C]). Note that V-A15 forms very large cells at 40°C (Phase, all panels shown at same magnification) and that nuclear DNA (DAPI) is not or unequally segregated resulting in large (double arrowhead) and small nuclei or nuclear fragments (arrowheads). Nuclear size was quantified by image analysis and is shown in (D); note wider size distribution at the restrictive temperature. (B) Immunofluorescence analysis of V-A15 using an antibody to MORN1 [20]. At the permissive temperature a clearly defined centrocone can be identified in each nucleus (Merge, arrow), at the restrictive temperature this organization is lost. (E and F) DNA content was also quantified by flow cytometry (see Figure 2 for experimental details). Distinct populations with DNA contents of <1N and >2N arise at the restrictive temperature (F). Note that flow cytometry might not detect very large parasites due to a filtering step, and that image analysis (D) will likely underestimate the number of <1N cells.