TheGiardialamellipodium-like ventrolateral flange supports attachment and rapid cytokinesis

Abstract

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The microtubule cytoskeleton plays a well characterized role in attachment via the ventral adhesive disc, whereas the role of the unconventional actin cytoskeleton is controversial. We identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin and is enriched in the ventrolateral flange (VLF), a lamellipodium-like membrane protrusion at the interface between parasites and attached surfaces. Live imaging revealed that the VLF grows to ~1 μm in width after cytokinesis, then remains size-uniform in interphase, grows during mitosis, and is resorbed during cytokinesis. A Flangin truncation mutant stabilizes the VLF and blocks cytokinesis, indicating that the VLF is a membrane reservoir supporting rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions, GlRac, the sole Rho family GTPase in Giardia, was localized to the VLF. Knockdown of Flangin, actin, and GlRac result in VLF formation defects indicating a conserved role for GlRac and actin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites challenged with fluid shear force in flow chambers had a reduced ability to remain attached, indicating a role for the VLF in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that is particularly important during mitosis when the parental ventral disc begins disassembly in preparation for cytokinesis.ImportanceThe ventrolateral flange (VLF) is a lamellipodium-like structure found at the host-parasite interface that has long been thought to be involved in parasite attachment. The proteins responsible for building the VLF have remained unidentified precluding manipulation of the VLF to determine its role in Giardia biology. We identified Flangin, a novel actin associated protein that localizes to the VLF, implicating Giardia actin in VLF formation. We demonstrate that: 1.) Flangin, actin, and GlRac are required for VLF formation, 2.) the VLF serves as a membrane reservoir to support Giardia’s incredibly fast cytokinesis, and 3) the VLF augments attachment, which is critical to parasitism. The microtubule-based adhesive ventral disc and the actin-based ventrolateral flange represent redundant means of maintaining attachment, the presence of redundant systems illustrate the importance of attachment to the lifestyle of this ubiquitous parasite.

Identification of Actin Filament Interactors in Giardia lamblia

“…Short GlActin filaments appear in the VLF, and another novel actin interactor, Flangin, has been implicated in its function (32); it is likely that more of these interactors are important for maintenance and function of the VLF.…”

“…In this case we are confident the protein is in the ER, as transmembrane proteins are necessarily trafficked through this organelle. Both GL50803_14551 (alpha-6 giardin) and GL50803_17255 (hypothetical) appeared in the ventrolateral flange, a thin membranous structure that protrudes from the plasma membrane and contributes to parasite attachment (32,33). GL50803_23833 (VPS35), GL50803_103855 (VPS29), GL50803_8560 (hypothetical), GL50803_15591 (coil coil protein), GL50803_4259 (clathrin light chain), and GL50803_11684 (leucine rich repeat protein) all had a punctate localization at the cell cortex, likely in association with the plasma membrane.…”

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

“…An additional explanation for the severity of attachment defects in Gl Actin-depleted cells is due to its involvements in supplemental mechanisms of attachment. Gl Actin has been demonstrated to function in building the ventrolateral flange, Giardia’s membrane lamella which also contributes to attachment [56,57]. Further, the marginal plate, a rigid structure associated with the anterior axonemes, is also hypothesized to be involved in attachment [10,30].…”

Staging Encystation Progression in Giardia lamblia Using Encystation-Specific Vesicle Morphology and Associating Molecular Markers

“…We were not surprised to find many statistically significant interacting proteins since GlRac, as the sole Rho GTPase in Giardia, is presumably responsible for many of the same roles the multitude of Rho GTPases carry out in other eukaryotes (Hodge and Ridley, 2016;Lawson and Ridley, 2018;Phuyal and Farhan, 2019). In addition to its role in membrane trafficking, we have shown that GlRac has important roles in regulating the cytoskeleton (Paredez et al, 2011;Krtková et al, 2017;Hardin et al, 2021); thus we expect many of the statistically significant interactors in non-encysting cells will prove to have roles in regulating the cytoskeleton. Here, we chose to focus on proteins predicted to have a role in trafficking as we had the potential to identify novel ESV components.…”

“…Note, constructs made to tag Giardia Rabs and putative GlRac-interactors were designed for stable integration by homologous recombination into the Giardia genome for endogenous expression. Generation of the native promoter morpholino-sensitive (ms) HALO_C18_GlRac_PuroR (designed for endogenous expression) and PAK promoter_CRIB_N11_mNG_3HA_NeoR (designed for episomal expression) constructs have been described previously (Hardin et al, 2021). Plasmid backbones used to build constructs in this paper were sourced from Gourguechon and Cande (2011); Krtková et al (2016), Michaels et al (2020), andParedez et al (2014).…”