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First published online 19 March 2009
doi: 10.1242/jcs.045740

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Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography

¹ Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
² Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA

View larger version (91K): [in this window] [in a new window]   Fig. 1. Tomographic representation. (A) Surface rendering representation of tomo 1, colours were subjectively attributed to organelles and structures around the flagellar pocket area (supplementary material Table S1). (B-D) Individual (sequential but not adjacent) tomographic slices (1.6 nm thick) extracted from the 435 slices that comprise the reconstructed 3D volume of tomo 1. (B) A slice of the actual tomogram at the level where the flagellum emerges from the flagellar pocket. (C) A slice at the level of the basal body (arrow) and probasal body (arrowhead). (D) A slice at the lower region of the pocket also showing the kinetoplast (arrow) within the mitochondrion. Scale bars: 200 nm in B-D. Movie 1 (supplementary material) shows the original tomographic reconstruction coupled with the segmentation model.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Flagellar pocket architecture. (A) Scanning electron micrograph illustrates the position of the flagellar pocket region. The flagellum exit point on the cell surface is labelled with an asterisk. (B) 3D model illustrates the relationship of the cytoskeletal and membrane structures associated with the pocket. Abbreviations: BB, basal body; PBB, probasal body; FP, flagellar pocket; PFR, paraflagellar rod; MtQ, microtubule quartet; FAZ, flagellum attachment zone; ER, endoplasmic reticulum. (C) This cartoon defines the axes that we used to position tomograms. The origin point is defined by the centre of the basal body at its most proximal end. The z-axis runs up the length of the axoneme; the x-axis is defined by the plane of the central pair microtubules at the point at which they are nucleated; finally, the y-axis points towards the probasal body. (D) A model of tomo 1 with many of the components excluded has been orientated such that the view is along the z-axis. This allows the definition of four quadrants in the cell useful for positioning organelles and structures and comparison of tomograms. Scale bars: 200 nm.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Flagellar pocket boundaries. (A) Representative thin-section electron micrograph illustrating the asymmetry of the flagellar pocket (FP) volume. Note the bulge side towards the cell interior accompanied by the probasal body (PBB). Note also a line of symmetry between the PBB and the single Golgi stack. (B) Model of tomo 1 showing only major membrane and cytoskeletal areas. Two boundaries define the flagellar pocket: the collar and the neck region defining the exit boundary, the radial fibres and collarette defining the flagellum entry boundary. BB, basal body. (C) Representative thin-section electron micrograph illustrating the boundaries that outline the flagellar pocket. Rectangles ouline the exit and entry boundaries shown at higher magnification in D and E, respectively. (F) Representative cross-section depicting the electron-dense collar (arrowheads) on the cytoplasmic side of the neck region membrane. (G,H) Views of collar density modelled from two tomograms showing a level of variation. The graphical representation of the collar shown here corresponds to the general electron-density patterns (arrowheads) seen in F. MtQ, microtubule quartet. (I) A view of the model of tomo 1 to illustrate the vase-like neck region through which the flagellum finally emerges from the cell. (J) Another view of the tomogram model to illustrate the neck region and flagellum exit point with a region of the cell surface membrane and associated subpellicullar microtubules in place. Scale bars: 200 nm.

View larger version (142K): [in this window] [in a new window]   Fig. 4. Basal bodies and flagellum entry boundary. (A) Representative cross-section of the transitional fibres radiating out from the basal body doublet microtubule region. (B) Representative cross-section of the collarette surrounding the flagellar membrane base. Black arrowheads denote the double tubular structures opposite each microtubule doublet. White arrowheads denote the linking fibrous sheath connecting these double tubular structures. (C) A model of tomo 1 showing the position of the radial fibres (arrowheads) located between the basal body and the surrounding membrane. (D) The sum of five individual tomographic slices (corresponding to a thickness of 8.5 nm) from a tomogram, illustrating the structure of a single radial fibre (arrowheads). (E) Thin section illustrating the cartwheel of the probasal body and striated fibre connections between probasal body and basal body (arrowheads). (F) Thin section of a 9+0 triplet microtubule basal body, illustrating a striated fibre extending from a C-tubule (arrowhead). (G) Thin section of a basal body and probasal body illustrating connections between each other and the microtubule quartet (MtQ) (arrowheads). (H) Electron micrograph of a detergent-extracted, salt-treated and negatively stained preparation of an isolated flagellum complex. Striated fibres have maintained the connections between the probasal body, basal body and microtubule quartet. Also seen in this micrograph are the microtubule quartet and the collar central annulus (arrowhead). PBB, probasal body. Scale bars: 200 nm.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Cytoskeletal structure. (A) Tomo 1 model view in which all membrane objects were excluded in order to emphasise the cytoskeletal elements around the flagellar pocket. Note the left-handed helical pattern of the microtubule quartet (MtQ), the position of the collar relative to the axoneme and the origin points of the flagellum attachment zone (FAZ) filament structure and paraflagellar rod (PFR). The origin of the microtubule quartet is indicated by an arrowhead. Scale bar: 200 nm. # indicates where both ends of the neck microtubule can be seen within the tomogram volume. (B) Representative cross-section of the flagellum (post-exit point) and its association to the cell body. The microtubule quartet (MtQ) and associated ER are seen to the left of the FAZ filament structure (when viewed from the posterior end of the cell). Scale bar: 200 nm. (C) Tomo 1 model view (observed from the anterior end of the cell) showing the MtQ and the FAZ filament structure on the neck region membrane. These structures then join the subpellicular microtubule array (SPMt). A neck microtubule is present on the neck region membrane on the other side of the FAZ filament structure. Scale bar: 200 nm. (D) General view of the subpellicular microtubule array within the tomographic volume of tomo 1. Microtubule ends within the tomographic volume are marked with # or an arrowhead. Plus ends of microtubules will be at the posterior end of the cell (left-hand side of this image). The arrowheads define the ending of three microtubules that open up the space required for the exit of the flagellum. Plus and minus ends of some subpellicular microtubules in which both ends could be seen are enlarged in E. Scale bar: 200 nm. (E) The tomographic volume of tomo 1 was rotated and sliced in order to bring the image of two microtubule ends into view (arrowheads). Plus and minus endings of the microtubules leave an enhanced gap between the microtubules for a short distance as illustrated. Note also the regularly arranged cross bridges between microtubules. Scale bars: 50 nm

View larger version (49K): [in this window] [in a new window]   Fig. 6. ER, Golgi and kinetoplast. These model views were chosen from the surface rendered representation of tomo 1 to provide a 3D impression of the complex endoplasmic reticulum (ER) architecture around the flagellar pocket. (A) A region of ER forms a sheet, closely adjacent to and covering the flagellar pocket membrane, that is continuous with an area of fenestration. (B) The model view in A was tilted 180° to show here the absence of ER lamina on the opposite side of the flagellar pocket. (C) The model view in A was tilted 90° around the z-axis to show here the Golgi localization relative to the ER and neck region: the single Golgi stack is adjacent to the fenestrated region of ER, following the line of symmetry with the probasal body and the bulge side of the flagellar pocket (see also Fig. 2A). The pink spheres represent individual ribosomes. (D) The kinetoplast region of the mitochondrion is illustrated in this model view. Scale bars: 200 nm.

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