Identification of Actin Filament Interactors in Giardia lamblia

Abstract

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.

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

“…We next asked if GL50803_16844, previously identified by mass spectrometry as a potential actin filament interactor at the ventral disc [17], could be involved in attachment. Hereafter, we will refer to this protein that lacks identifiable domains and homologues in other organisms as Disc- and Actin-Associated Protein 1 (DAAP1).…”

“…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. Another explanation is that Gl Actin works to stabilize one of the non-microtubule components of the ventral disc.…”

“…In contrast, depletion of median body protein with morpholinos results in broken discs [11]. Although an inability to attach can be attributed to disc defects [11,28], the depletion of gamma-giardin, another disc-localized protein that interacts with Gl Actin [17], resulted in malformed discs with normal attachment [55]. These findings indicate that some disc proteins may be involved in maintaining the structural integrity of the disc, while others are involved in controlling conformational changes of the disc required for modulating attachment.…”

“…However, since over 25% of our unattached Gl Actin-depleted cells had apparently normal discs, the attachment defect cannot be attributed to disc defects alone. Our previous study identified eight other putative disc-localized F- Gl Actin interactors which could also contribute to attachment and/or disc morphology [17].…”

“…We recently identified eight Giardia actin ( Gl Actin) interactors that localize to the ventral disc [17], suggesting a means to resolve the role of actin and actin-binding proteins in ventral disc function. One such actin-interacting protein, GL50803_16844, which we denote Disc and Actin Associated Protein 1 (DAAP1), appears highly enriched in the two regions of the disc important for fluid flow during attachment.…”