Identification of Actin Filament-Associated Proteins in Giardia lamblia

Steele-Ogus, Melissa C.; Johnson, Richard S.; MacCoss, Michael J.; Paredez, Alexander R.

doi:10.1128/spectrum.00558-21

Cited by 5 publications

(6 citation statements)

References 81 publications

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“…The ventral disc is well known as a Giardia- specific microtubule-based structure [ 16 , 31 ] and our data show that the actin cytoskeleton has an important role in ventral disc assembly and function. A functional relationship between actin and microtubules has been well-documented for other structures.…”

Section: Discussionsupporting

confidence: 57%

“…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 , 15 , 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%

“…Only three DAPs identified to date contain known microtubule-binding motifs; most are ankyrin-repeat proteins, NEK kinases, or the annexin-like alpha-Giardins [ 9 , 15 ]. At least 25, however, lack identifiable domains and are annotated in the Giardia genome as “hypothetical proteins” [ 16 , 17 ]. So far, no DAPs have been identified as motor or force-generating proteins.…”

Section: Introductionmentioning

confidence: 99%

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Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia

et al. 2022

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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.

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Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia

et al. 2022

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“…Therapeutic strategies targeting attachment would therefore effectively clear infection. The results presented here in combination with our recent study of Gl Actin’s role in ventral disc function indicate that Giardia’s highly divergent actin homolog has multiple roles in supporting attachment [ 5 , 6 ]. Inhibiting Gl Actin would simultaneously disrupt ventral disc and ventrolateral flange function, making it a promising therapeutic target.…”

Section: Discussionsupporting

confidence: 56%

“…The prominent microtubule based ventral adhesive disc plays a central role in supporting parasitic attachment [ 4 ]. Recently we used proteomics to identify actin binding proteins in Giardia and found several that localized to portions of the ventral disc which indicates that the actin cytoskeleton also plays a role in parasitic attachment [ 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

et al. 2022

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Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.

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