A Trypanosoma brucei protein required for maintenance of the flagellum attachment zone and flagellar pocket ER domains.
We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket.The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket.Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
- Protozoan Proteins/metabolism*
- Trypanosoma brucei brucei/physiology*/ultrastructure
- Amino Acid Sequence
- Cytoskeletal Proteins/metabolism/ultrastructure
- Electron Microscope Tomography
- Endoplasmic Reticulum/metabolism/ultrastructure
- Gene Knockdown Techniques
- Imaging, Three-Dimensional
- Membrane Proteins/metabolism
- Models, Molecular
- Molecular Sequence Data
- RNA Interference
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fig0015: EM tomography analysis of the FAZ ER. A) Illustration of the FAZ as viewed in longitudinal 3D models (left, adapted from Lacomble et al., 2009a) or by cross-sections of TEM (right). On the top left of the tomography image, a scanning electron micrograph of a procyclic trypanosome (scale bar, 1 μm) shows the area covered by the 3D model. B) Slice of a tomogram through the FAZ area immediately anterior to the flagellar pocket, viewed from the posterior of the cell. Next to the flagellar axoneme (Ax), the paraflagellar rod (PFR) is linked to the membrane at the point of contact between the flagellum and the cell body (arrowhead). In the cytoplasm immediately to the left of this point, the 4 microtubules of the MtQ are interdigitated by the FAZ ER, which is connected to a section of central ER (arrows). Scale bar, 100 nm. C) Tomographic reconstruction of the FAZ ER and neighbouring structures in the area boxed in A. Upon reconstruction, the two junctions between the FAZ ER and the central ER appear as ‘tube-like’ membrane bridges (arrows). D) Slice of a tomogram in the FAZ area (likely anterior to the nucleus), showing ribosomes associated with the FAZ ER (arrows), indistinguishable from those found in a neighbouring section of rough ER (arrowheads). Scale bar, 50 nm. E) 3D reconstruction of the FAZ ER from the area in C, showing associated ribosomes (purple spheres) and a clear tube-like bridge linking it to the rough ER (arrow). In this tomogram, only 3 of the 4 MtQ microtubules appeared encircled by ER membranes, and a “reduced microtubule” (arrowhead) appeared as a thin rod immediately to the right of the MtQ. F) TEM cross-section through the FAZ showing 3 MtQ microtubules with associated FAZ ER (arrowheads) and the “reduced microtubule” to the right of the MtQ (arrow). Scale bar, 100 nm. G) Larger magnification of a section of E highlighting the membranous nature of the “reduced microtubule” (arrow), which will be referred to henceforth as ‘FAZ tubule‘. Note the similarity between the walls of the FAZ tubule and the membrane of the FAZ ER nearby (arrowhead). Scale bar, 50 nm. Colour scheme in C and E: subpellicular microtubules, light blue; MtQ microtubules, dark blue; ER, green; ribosomes, purple; Golgi complex, orange; endosomes, red; plasma membrane, grey; “reduced microtubule” or FAZ tubule, yellow.
In order to clarify the morphology and nature of the FAZ ER, we performed 3D reconstructions of the FAZ ER by electron microscopy tomography in the bloodstream form of T. brucei (Fig. 1, and Supplementary Material Movies S1a, S1b, S2a and S2b). We observed connections between the FAZ ER and the central ER at different positions along the length of the FAZ. Data from two representative sections along the major axis of the cell are shown here. The section in Figure 1 (B and C), and movies S1a and S1b corresponds to an area immediately after the flagellum emerges from the flagellar pocket, identified by the presence of the Golgi complex. The section shown in Figure 1 (D and E) and movies S2a and S2b is from a different area along the major axis of the cell, most likely between the nucleus and the anterior end, since it shows the FAZ but does not include either the Golgi or the nucleus.