An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasiteToxoplasma gondii

Abstract

The human parasite Toxoplasma gondii has 22 regularly spaced microtubules associated with the cortex. This work defines the differential localization of associated proteins, explores the biophysical constraints on specific targeting along the cortical microtubules, and investigates the function of these proteins in stabilizing the polymers.

Loss of a doublecortin (DCX)-domain protein causes structural defects in a tubulin-based organelle of Toxoplasma gondii and impairs host-cell invasion

“…, 2008). With the advantage of SIM, that spot of tubulin can now be resolved into a five-petaled flower-like arrangement (Figure 4), as previously observed (Liu et al. , 2016).…”

Stability and function of a putative microtubule-organizing center in the human parasiteToxoplasma gondii

“…, 2010; Liu et al. , 2013, 2016), and knock-in and knockout clones were confirmed by Southern blotting analysis (Figure 5B). The Δapr1 parasites formed noticeably smaller plaques than either the wild-type or the parental APR1-mCherryFP knock-in parasites (Figure 5C).…”

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

“…Microtubules are also assembled into the mitotic spindle during parasite replication. Nascent daughter buds are formed close to the poles of the mitotic spindle, with each pole resembling a “comet tail” that marks the basal end of the daughters growing within the mother parasite [13]. Individual parasites within an intracellular eight-parasite vacuole were visible in the raw data collected by objective C, but out-of-focus haze obscured the cortical microtubules, and poor axial resolution distorted the cross-sectional parasite shapes [Figs.…”

“…gondii β 1-tubulin isoform fused to EGFP, and driven by the α 1-tubulin promoter [10], were used. Parasite cultures were maintained by serial passage in confluent primary human foreskin fibroblast (HFF, ATCC# SCRC-1041) monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) lacking phenol red and antibiotics, supplemented with 4.5 g/L D-glucose, 1% (v/v) heat-inactivated cosmic calf serum (Hyclone), 1 mM sodium pyruvate (Gibco), and 2 mM GlutaMAX-I L-alanyl L-glutamine dipeptide (Gibco), as previously described [13,29]. To prepare the imaging samples, HFFs were seeded onto cleaned, sterile 24 × 50 mm #1.5 glass coverslips to obtain 70%–80% confluency, inoculated with the EGFP-Tg β 1-tubulin-expressing parasites described above, and incubated at 37°C with 5% CO 2 for 19–20 h. The T .…”