Tomographic reconstructions of the endosomal apparatus of T. brucei after cargo uptake reveals a large and mostly continuous membrane system.

(A) Elongated and highly fenestrated endosomal sheets and palisades. (B) Elongated and slightly fenestrated endosomes. (C) Large circular endosomal cisternae. (D) Two different substructures are displayed: one contains tubular palisades (I) while the other one is a heavily fenestrated sheet (II). HRP endocytosis and DAB photooxidation were performed prior to tomogram acquisition (A, B and D). Ferritin endocytosis was performed prior to tomogram acquisition (C). All sections are 250 nm thick. The reconstructions (A) and (D) are based on three and two sections, respectively. The reconstructions (C) and (D) are based on one section each. The section and corresponding tomogram (C) originate from the same sample block as the image in Figure 4 I from Engstler et al. (2004). Endosomal membranes are shown in green. The endoplasmic reticulum is visualised in white colour. The Golgi apparatus is labelled in orange and the mitochondrion is shown in red. Abbreviation: flagellar pocket (FP). Scale bars: 500 nm. Movies corresponding to panel A-D can be found here: A) z-stack, 3D model; B) z-stack, 3D model; C) z-stack, 3D model; D) z-stack, 3D model.

Super-resolution light microscopy reveals continuous endosomal membranes.

(A) Visualisation of the endosomal system in T. brucei by direct stochastic optical reconstruction microscopy (dSTORM). A transgenic EP1::HaloTag expressing bloodstream form cell line was generated and used to visualise the endosomal system. Cells were labelled with a HaloTag ligand and fixed with 4 % formaldehyde. Shown are a merge of transmitted light, DAPI staining and the EP1::HaloTag signal (I), a merge of DAPI staining and the EP1::HaloTag signal (II), the EP1::HaloTag signal (III) and a magnified view of the EP1::HaloTag signal (IV). (B) Visualisation of the endosomal system in T. brucei using expansion microscopy. Bloodstream form cells were pulsed with dextran conjugated to Alexa488 and fixed with 4 % formaldehyde and 0.2 % glutaraldehyde. Shown are exemplary images (I – IV) of not expanded and expanded cells imaged using widefield microscopy. All images represent a merge of DAPI staining (cyan) and dextran signal (magenta). Flagellar pockets (FP) are annotated. (C) 3D presentation of the endosomal system using a combination of expansion microscopy and structured illumination microscopy (SIM). The same gels as imaged in (B) were analysed with SIM. The 3D presentation was generated using the IMARIS package (Bitplane). (D) Scatter plot of the quantification of the expansion factor. The diameter of the fluorescence signal corresponding to flagellar pocket (FP) and length of the endosomes were measured in multiple not expanded (no ExM) and expanded cells (ExM) (n ≥ 15). The median values are indicated as red bars and the corresponding numbers are shown in the upper part of the panel.

The endosomal markers TbRab5A, TbRab7, and TbRab11 partly colocalize in the posterior region of the cell.

(A) The experimental workflow, from fluorescence images to object generation and quantitative 3D colocalization analysis, is exemplified. EP1::GFP (I) expressing cells were fixed and labelled with anti-TbRab5A (II), anti-TbRab7 (III) and anti-TbRab11 (IV) antibodies and imaged using widefield microscopy. Upper row: maximum intensity projections (MIP) for each individual fluorescence channel (I – IV) as well as a merge of all three anti-TbRab marker channels (V). The outline of the cell is shown by a dashed line (I - V) and the region of interest (endosomal system) is presented by a solid line (I - V). Middle row: Corresponding 3D objects to display the region of interest (VI) and the TbRab marker objects (VII – IX). The merge of all objects confirmed that the TbRab marker objects are located within the region of interest (X). Lower row: Colocalization of the objects representing TbRab5A and TbRab7 (XI), TbRab5A and TbRab11 (XII), TbRab7 and TbRab11 (XIII). The x-, y- and z-axes are indicated to support the three-dimensional view. (B) Object colocalization shown for additional cells. The colocalization of TbRab5A and TbRab7 (I), TbRab5A and TbRab11 (II), and TbRab7 and TbRab11 (III) is shown for different cells. (Cells 1 – 4). (C - D) Quantification of colocalization analysis for TbRab marker combinations. The colocalization function of IMARIS was used for the quantification. The EP1 surfaces defined the ROI (see panel A) and the automatic thresholding function (Costes et al., 2004) ensured minimal user bias. Each data point (n = 38) represents a field of view with 30 – 40 cells, corresponding to a total number of ∼ 1,200 analysed cells. The median is highlighted with a red line and the corresponding number is written on top of the dataset. (C) Scatter plot of the colocalization analysis for the different TbRab marker combinations using the percentage of volume overlap. (D) Scatter plot of the colocalization analysis for the different TbRab marker combinations using the Pearson correlation coefficient.

The endosomal markers TbRab5A and TbRab7 are present on the same membrane.

(A) Schematic representation of antibody binding to visualise the maximum linkage error of 30 nm between a bound epitope and the gold particle. (B - G) Electron micrographs of cryosections labelled with rat anti-TbRab5A and guinea pig anti-TbRab7 antibodies and visualized with 6 or 12 nm gold-coupled secondary antibodies. For each panel an annotated and an unedited version of the image is presented. Endosomes are highlighted in green. Gold particles corresponding to TbRab5A signals are labelled in yellow. Gold particles corresponding to TbRab7 signals are highlighted in cyan. Scale bars: 200 nm.

The endosomal markers TbRab5A and TbRab11 are present on the same membranes.

(A - F) Electron micrographs of cryosections labelled with rat anti-TbRab5A and rabbit anti-TbRab11 antibodies and visualized with 6 or 12 nm gold-coupled secondary antibodies. For each panel an annotated and an unedited version of the image is presented. Endosomes are marked in green, and the lysosome is highlighted in blue. Gold particles are marked in yellow (TbRab5A) and in magenta (TbRab11). Scale bars: 200 nm. Exocytic carrier (EXC), lysosome (L), plasma membrane (PM).

The endosomal markers TbRab7 and TbRab11 are present on the same membranes.

(A - G) Electron micrographs of cryosections labelled with guinea pig anti-TbRab7 and rabbit anti-TbRab11 antibodies visualized with 6 or 12 nm gold-coupled secondary antibodies. For each panel an annotated and an unedited version of the image is presented. Endosomes are highlighted in green. Gold particles are labelled in cyan (TbRab7) and in magenta (TbRab11). Scale bars: 200 nm.

Cargo uptake and anti-VSG immunogold labelling confirm the endosomal identity.

(A – I) Electron micrographs of cryosections labelled with anti-TbRab5A (yellow), anti-TbRab7 (cyan) or anti-TbRab11 (magenta) antibodies and 12 nm gold-conjugated secondary antibodies. For each panel an annotated and an unedited version of the image is presented. Endosomes are highlighted in green, the lysosome in blue. Parasites were incubated with 5 nm gold-conjugated BSA (A – C) or transferrin (Tf) (D – F), prior to fixation, sectioning and immunolabelling. BSA and transferrin cargo was observed in vesicles, endosomes, and lysosome. (H – I) Cells were labelled with anti-VSG antibodies and 6 nm gold-conjugated secondary antibodies. (F), flagellum, (FP) flagellar pocket, (PM) plasma membrane, (L) lysosome. Scale bars: 200 nm.

3D Tokuyasu confirmed continuous endosomal membranes in cryosections.

(A, B) Tomographic reconstruction of the endosomal apparatus. (I – III) Isolated view of different organelle structures. Cryosections (250 nm) were labelled with anti-TbRab antibodies and 6 or 12 nm gold-coupled secondary antibodies. Endosomes are shown in green, lysosomes in blue, the flagellar pocket in white, the Golgi apparatus in orange. Gold particles are indicated as coloured spheres; TbRab5A (yellow), TbRab7 (cyan) or TbRab11 (magenta). Scale bars: 500 nm. Movies corresponding to panel A and B can be found here: A) z-stack, 3D model; B) z-stack, 3D model.

Schematic representation of the endosomal system in T. brucei.

The endosome is marked by the presence of small GTPases of the Rab family: Rab5A (yellow dots), Rab7 (cyan dots), and Rab11 (red dots). Class I clathrin-coated vesicles (CCV-I), class II clathrin-coated vesicles (CCV-II), vesicles coated with coat protein (COP) II.