Cellular landmarks of Trypanosoma brucei and Leishmania mexicana

Abstract

The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.

Graphical abstract

Fig. 1

The morphology of T. brucei and L. mexicana. A–C. The morphologies of key culturable life cycle stages of T. brucei and L. mexicana, shown in cartoon form (Left) and as an overlay of a phase contrast and Hoechst (DNA stain) fluorescence micrographs (Right). A. Procyclic form T. brucei with a trypomastigote morphology. The anterior-posterior axis, the kinetoplast (K) and nucleus (N), the flagellum proximal-distal axis and the dorsal-ventral axis are indicated. B. Procyclic form L. mexicana with a promastigote morphology. No features visible by light microscopy can be used to define a dorsal-ventral axis. C. Amastigote form L. mexicana. The flagellum does not protrude from the cell, meaning a flagellum proximal-distal axis is not easy to identify. D–E. The key cell cycle stages of procyclic form T. brucei and L. mexicana, showing the order of duplication of the kinetoplast (K), nucleus (N) and flagellum (F) and their morphology. D. Procyclic trypomastigote form T. brucei. E. Procyclic promastigote form L. mexicana.

Fig. 2

Reference protein localisations for procyclic trypomastigote form T. brucei. Widefield fluorescence images for each protein are laid out in the same format: Left, an overlay of the phase contrast (grey), mNG fluorescence (green) and Hoechst DNA stain (magenta) and right, the mNG fluorescence in greyscale. These images were all captured as part of the TrypTag project. The protein name and gene fusion are shown in the top left (Tb927.X.XXXX::mNG for C terminal tagging, mNG::Tb927.X.XXXX for N terminal tagging). The annotation of the localisation is shown in the bottom left. A key distinguishing feature of the localisation may be highlighted on the right.

Fig. 3

Reference protein localisations for promastigote procyclic L. mexicana. Widefield fluorescence images of the localisation of the L. mexicana orthologs of the proteins shown in Fig. 2. Localisations are presented in the same order and using the same layout as for T. brucei for easy comparison.

Fig. 4

Reference protein localisations for axenic amastigotes L. mexicana. Widefield fluorescence images of the localisation of the L. mexicana proteins shown in Fig. 3. Localisations are presented in essentially the same order and using the same layout as Fig. 2, Fig. 3. Image contrast for fusion proteins not expressed in the amastigote approximately matches the contrast in Fig. 2. Red outlines indicate a localisation that may be spurious, see main text for more detail.

Fig. 5

Cell cycle dependent localisations in T. brucei and L. mexicana. Widefield fluorescence images of key cell cycle dependent localisations in T. brucei procyclic trypomastigotes and L. mexicana procyclic promastigotes and amastigotes. For each protein localisation the number of kinetoplasts (K), nuclei (N) and (where visible) flagella (F) are indicated. Image contrast is the same for each cell cycle stage. L. mexicana amastigotes had few dividing cells after 72 h differentiation, so the first amastigote morphology division (10 h after division) is shown.