A quorum sensing-independent path to stumpy development in Trypanosoma brucei
Fig 5
Ectopic VSG overexpression-induced ES-attenuation causes stumpy development.
The analyses were conducted with an arrested clone of the GFP:PAD1UTR reporter cell line at densities below 5x 105 cells/ml (except for the density-induced control). (A) The cell cycle position of DAPI-stained trypanosomes was analyzed after 24 and 48 hours of tetracycline induction. Non-induced slender (0 h) and density-induced stumpy cells (st) served as controls. The configuration of the kinetoplast (K) and nucleus (N) was microscopically analyzed. Only cells with a non-dividing kinetoplast and a single nucleus are in the G1-phase (1K1N). Parasites with a dividing kinetoplast (1Kd1N) are in the mitochondrial S-phase. Trypanosomes with two kinetoplasts (2K1N) are in G2/M-phase, while parasites with two kinetoplasts and nuclei (2K2N) are post mitotic. Parasites with an abnormal K/N configuration were termed "other". Values are given as percentages (± SD) of two experiments (total n > 500). (B) Representative microscopic image of cells ectopically overexpressing VSG 121 for 48 hours. In the left image DAPI (grey) and GFP:PAD1UTR fluorescent signals (green) illustrate the 1K1N cell cycle arrest and the expression of the stumpy stage reporter. The DIC image on the right documents the short stumpy morphology of the trypanosomes. Note the characteristic short free part of the flagellum (white arrowhead). Scale bar: 10 μm. (C) Western blot stained with an antibody against the mitochondrial lipoamide dehydrogenase (LipDH, green), whose expression increases during stumpy development. This shows that LipDH is up-regulated during ectopic VSG overexpression-induced ES-attenuation. Detection of paraflagellar rod (PFR) proteins served as a loading control (magenta). (D) Deconvolved three-channel 3D images of chemically fixed non-induced slender cells (0 h, upper panel) and parasites ectopically overexpressing VSG 121 for 48 hours (48 h, lower panel). The mitochondrion was stained with mitotracker (magenta), the GFP:PAD1UTR reporter is shown in green and DAPI in grey. The upper panels show the shape of typical elongated mitochondria of slender cells, and the lower ones branched stumpy-like mitochondria of ectopic VSG overexpressors (48 h). Scale bars: 5 μm.