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

Abstract

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.

ImagingGiardia intestinaliscellular organisation using expansion microscopy revealed atypical centrin localisation

“…Physical expansion combined with post-expansion labelling and confocal microscopy increased the resolution of resulting images by the factor of 3.4, which is slightly less than the usual expansion index of 4.5 (Wassie et al 2019), however we demonstrate that it clearly enables resolving of details previously not accessible by conventional light microscopy. The post-expansion labelling generally improves the epitope accessibility of expanded samples and reduces antibody linkage errors proportionally to the expansion factor (Sauer 2021), and was therefore preferred in our experiments over pre-expansion labelling previously used in Giardia (Hardin et al 2019(Hardin et al , 2022. Our protocol utilizes a conventional confocal microscope routinely present in microscopy facilities instead of less common superresolution devices.…”

“…Parasitic protists are a medically and veterinary important group of eukaryotic microbes, and ExM has been successfully applied to Plasmodium, Trypanosoma, Leishmania, Toxoplasma and Trichomonas to reveal in great detail their cytoskeletal organisation and localisation of their cytoskeleton-associated proteins (Halpern et al 2017, Betriaux et al 2021, Gorilák et al 2021, Kalichava and Ochsenreiter 2021, Dos Santos Pacheco et al 2022, Bandeira et al 2023). Although this method has already been applied also to Giardia in a combination with superresolution microscopy (Halpern et al 2018, Hardin et al 2022), here, we present a workflow that does not require a superresolution microscopy equipment. It is based on a robust ultra-structure expansion microscopy, which is commonly used for imaging of cytoskeletal structures (Gambarotto et al 2021) and on a modification of the method in terms of effectiveness and epitope accessibility via postexpansion labelling.…”

Functional Differentiation of Cyclins and Cyclin-Dependent Kinases in Giardia lamblia

“…To understand the GlCDK- and Glcyclin-mediated processes occurring in Giardia in vivo , observations of the live cells are essential to validate the results obtained from the experiments on the fixed cells. Live imaging of Giardia cells had been successfully performed in elaborate studies of biogenesis of the mitosome ( 31 ), flagellar length control ( 32 ), the role of never-in-mitosis A-related kinase 8455 (Nek8455) in cell division ( 33 ), identification of noncanonical actin-binding proteins ( 34 ), and involvement of the ventrolateral flange in Giardia attachment ( 35 ). The next step we should achieve is to establish the tool for live imaging of Giardia cells to confirm the data derived from the fixed cells.…”

A novel mechanism of microbial attachment: the flagellar pump ofGiardia lamblia

“…The spiral microtubule array that forms the ventral disk is prominent (although the dome-like morphology is not evident in the twodimensional image). The two flagella are confined by the ventral groove throughout the beating period, and while not evident in the optical images, electron microscopy images show that the flagella are tightly confined on the dorsal side by the ventral groove (8,14,15). Aided by the fin-like projection on the ventral side (Fig.…”