The SUN-like protein TgSLP1 is essential for nuclear division in the apicomplexan parasite Toxoplasma gondii

Authors:
Mirjam Wagner, Yuan Song, Elena Jiménez-Ruiz  , Sonja Härtle, Markus Meissner
In:
Source: J Cell Sci
Publication Date: (2023)
Issue: 136: 21
Research Area:
Parasitology
Cells used in publication:
Toxoplasma gondii
Species: unicellular
Tissue Origin:
Platform:
4D-Nucleofector® X-Unit
Experiment

To generate stable parasite lines, parasites were transfected using a P3 Primary cell 4D-Nucleofector X kit L, V4XP-3024 from Lonza. 106–107 freshly lysed RH-?ku80-DiCre parasites were centrifuged (1500 g for 5 min), resuspended in 100 µl P3 buffer, mixed with prepared DNA (see section, ‘Generation of tagged and floxed strains’) and electroporated within a 100 µl cuvette using the Amaxa 4D-Nucleofector system from Lonza. For electroporation, the programme FI-158 was used. Transfected parasites were resuspended in fresh DMEM and added onto confluent HFF cells.

Abstract

Connections between the nucleus and the cytoskeleton are important for positioning and division of the nucleus. In most eukaryotes, the linker of nucleoskeleton and cytoskeleton (LINC) complex spans the outer and inner nuclear membranes and connects the nucleus to the cytoskeleton. In opisthokonts, it is composed of Klarsicht, ANC-1 and Syne homology (KASH) domain proteins and Sad1 and UNC-84 (SUN) domain proteins. Given that the nucleus is positioned at the posterior pole of Toxoplasma gondii, we speculated that apicomplexan parasites must have a similar mechanism that integrates the nucleus and the cytoskeleton. Here, we identified three UNC family proteins in the genome of the apicomplexan parasite T. gondii. Whereas the UNC-50 protein TgUNC1 localised to the Golgi and appeared to be not essential for the parasite, the SUN domain protein TgSLP2 showed a diffuse pattern throughout the parasite. The second SUN domain protein, TgSLP1, was expressed in a cell cycle-dependent manner and was localised close to the mitotic spindle and, more detailed, at the kinetochore. We demonstrate that conditional knockout of TgSLP1 leads to failure of nuclear division and loss of centrocone integrity.