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The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

ABSTRACT: Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.

SUBMITTER: Woessner DJ

PROVIDER: S-EPMC3294447 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

Woessner David J DJ Dawson Scott C SC

Eukaryotic cell 20120113 3

Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ven ...[more]

PMID: 22247266

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Project description: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.

Project description:C8ORF37 is a causative gene for three different clinical forms of incurable retinal degeneration. However, the completely unknown function of C8ORF37 limits our understanding of the pathogenicity of C8ORF37 mutations. Here, we performed a comprehensive phenotypic characterization of a C8orf37 KO mouse line, generated using CRISPR/Cas9 technology. Both C8orf37 KO male and female mice exhibited progressive and simultaneous degeneration of rod and cone photoreceptors but no non-ocular phenotypes. The major ultrastructural feature of C8orf37 KO photoreceptors was massive disorganization of the outer segment (OS) membrane discs starting from the onset of disc morphogenesis during development. At the molecular level, the amounts of multiple OS-specific membrane proteins, including proteins involved in membrane disc organization, were reduced, although these proteins were targeted normally to the OS. Considering the distribution of C8ORF37 throughout the photoreceptor cell body, the normal structure of the KO photoreceptor connecting cilium, and the absence of defects in other ciliary organs of the KO mice, our findings do not support the previous notion that C8ORF37 was a ciliary protein. Because C8ORF37 is absent in the photoreceptor OS, C8ORF37 may participate in the secretory pathway of OS membrane proteins in the photoreceptor cell body and thus maintain the homeostasis of these proteins. This study established a valid animal model for future therapeutic studies of C8ORF37-associated retinal degeneration. This study also shed new light on the role of C8ORF37 in photoreceptors and on the pathogenic mechanism underlying retinal degeneration caused by C8ORF37 mutations.SIGNIFICANCE STATEMENT Inherited retinal degeneration is a group of incurable conditions with poorly understood underlying molecular mechanisms. We investigated C8ORF37, a causative gene for three retinal degenerative conditions: retinitis pigmentosa, cone-rod dystrophy, and Bardet-Biedl syndrome. C8ORF37 encodes a protein with no known functional domains and thus its biological function is unpredictable. We knocked out the C8ORF37 ortholog in mice, which resulted in a retinal phenotype similar to that observed in patients. We further demonstrated that C8ORF37 is required for photoreceptor outer segment disc formation and alignment, a process that is critical for photoreceptor function and survival. This study advances our understanding of the pathogenesis of retinal degeneration and establishes a valuable mouse model for future therapeutic development.

Dataset Information

The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

Publications

The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

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