Abstract
Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.
Highlights
Giardiasis is the most common cause of protozoan intestinal infection worldwide [1], and has been included in the World Health Organization (WHO) Neglected Diseases Initiative as one of a group of diseases of global importance that are linked with poverty and limit development and socio-economic improvements [1,2]
The type of structural data obtained in this study is comprised of A) an overview of the microtubule cytoskeleton of the whole organism (Figures 1A–1C) at,75 nm resolution, B) the arrangement of the ventral disc microtubules/microribbons using conventional electron tomography (Figures 1D–1F) at,5 nm resolution, and C) unprecedented higher resolution (.3 nm) detail of microtubule and microribbon organization (Figures 2, 3, 4, 5, 6, 7) using volume-based averaging procedures on tomograms of frozen-hydrated specimens
We have obtained the first 3-D reconstruction of an entire Giardia lamblia trophozoite using a new procedure called 3ViewH [18] that has been developed by Gatan Inc. (Pleasanton, CA). 3ViewH combines sequential microtome sectioning with scanning electron microscopy back-scattered electron imaging of each fresh blockface (Figure 1)
Summary
Giardiasis is the most common cause of protozoan intestinal infection worldwide [1], and has been included in the World Health Organization (WHO) Neglected Diseases Initiative as one of a group of diseases of global importance that are linked with poverty and limit development and socio-economic improvements [1,2]. Beyond canonical axonemes and spindle assemblies, many parasitic protists have evolved elaborate microtubule-based arrays that enable specialized functions throughout their complex life cycles Each of these arrays have a unique supramolecular architecture, including the subpellicular microtubule array of apicomplexans [5] (e.g., the conoid of Toxoplasma [6]) and trypanosomes [7]. These elaborate structures are composed of numerous and novel microtubule-associated proteins (MAPs) that presumably enable the unique structural and dynamic functions of these microtubule-based arrays. Very few of these specialized microtubule assemblies have been imaged in three dimensions in situ, but understanding their organization in detail is critical toward ascertaining the function of these organelles in mediating the pathogenesis of the parasites
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