The ventral disc is a flexible microtubule organelle that depends on domed ultrastructure for functional attachment of<i>Giardia lamblia</i>

Dawson, Scott C.; Nosala, Christopher

doi:10.1101/213421

Cited by 7 publications

(12 citation statements)

References 23 publications

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“…It is noteworthy that the different disc proteins had different localizations, indicating that the different regions of the disc may have different relationships to GlActin. Perhaps most notable are GL50803_16844 and GL50803_113644, as they are highly enriched in the margin near the overlap zone and/or the ventral groove; these regions are hypothesized to regulate fluid flow beneath the ventral disc (69). While GlActin does not appear concentrated in the ventral disc in immunofluorescence images, this could be due to steric hindrance between the antibody and proteins on the ventral disc, similar to the faint tubulin immunostaining seen in the disc compared to the amount of signal observed when using fluorescentlytagged tubulin (36).…”

Section: Discussionmentioning

confidence: 99%

Identification of Actin Filament Interactors in Giardia lamblia

Steele-Ogus

Johnson

MacCoss

et al. 2021

Preprint

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The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of non-canonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin), rather than filamentous actin (F-actin). To identify interactors, we used a chemical crosslinker to preserve native interactions, followed by an anti-GlActin antibody, Protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched in the conditions favoring F-actin. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each category was co-localized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial.

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Section: Discussionmentioning

confidence: 99%

Identification of Actin Filament Interactors in Giardia lamblia

Steele-Ogus

Johnson

MacCoss

et al. 2021

Preprint

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show abstract

“…It is noteworthy that the different disc proteins had different localizations, indicating that the different regions of the disc may have different relationships to Gl Actin. Perhaps most notable are GL50803_16844 and GL50803_113644, as they are highly enriched in the margin near the overlap zone and/or the ventral groove; these regions are hypothesized to regulate fluid flow beneath the ventral disc ( 70 ). While Gl Actin does not appear concentrated in the ventral disc in immunofluorescence images, this could be due to steric hindrance between the antibody and proteins on the ventral disc, similar to the faint tubulin immunostaining seen in the disc compared to the amount of signal observed when using fluorescently tagged tubulin ( 7 ).…”

Section: Discussionmentioning

confidence: 99%

Identification of Actin Filament-Associated Proteins in Giardia lamblia

et al. 2021

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Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin ( Gl Actin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin.

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“…Another possibility is that Gl Actin helps maintain the structure of the disc, consistent with its role in maintaining cell and organelle shape in other organisms [18,21]. The unusual stability of disc microtubules can be at least partially attributed to certain DAPs [8,14,16], which lock the microtubules into place, preventing their turnover. Gl Actin interacts with at least one of these DAPs, GL50803_5188 [17], and could be working in conjunction with it to stabilize the disc microtubules themselves.…”

Section: Discussionmentioning

confidence: 99%

“…In the current model, both hydrodynamic flow and suction contribute to the mechanism of attachment [9]. Attachment occurs in several distinct stages [8,9]. In early attachment, the anterior ventrolateral flange, lateral crest and ventral groove make contact with the surface while the ventral flagella beat.…”

Section: Introductionmentioning

confidence: 99%

Disc and Actin-Associated Protein 1 Influence Attachment in the Intestinal Parasite Giardia lamblia

Steele-Ogus

Obenaus

Sniadecki

et al. 2021

Preprint

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The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment.Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.Author SummaryGiardia lamblia is a single-celled organism and one of the most common gastrointestinal parasites worldwide. In developing countries, recurrent Giardia infections are common, due to lack of access to clean water. Giardia infections can lead to diarrhea, vomiting, dehydration, disruption of the intestinal microbiome, and chronic infections can lead to colitis and irritable bowel syndrome. Because existing drug treatments have side effects and Giardia’s resistance to drugs is increasing, new treatment strategies are needed. The parasite’s attachment to the host’s intestine is mediated by a Giardia-specific structure that resembles a suction cup and is called the ventral adhesive disc. We previously identified DAAP1, a protein which interacts with Giardia actin and localizes to the ventral disc. Here, we explore the relationship between these two proteins and investigate their role in disc-based attachment. Most disc proteins, including DAAP1, are unrelated to any human proteins, making them appealing drug targets to inhibit parasite attachment and infection.

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The ventral disc is a flexible microtubule organelle that depends on domed ultrastructure for functional attachment ofGiardia lamblia

Cited by 7 publications

References 23 publications

Identification of Actin Filament Interactors in Giardia lamblia

Identification of Actin Filament Interactors in Giardia lamblia

Identification of Actin Filament-Associated Proteins in Giardia lamblia

Disc and Actin-Associated Protein 1 Influence Attachment in the Intestinal Parasite Giardia lamblia

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