Static Clathrin Assemblies at the Peripheral Vacuole—Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia

Abstract

Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain ( GlCHC), dynamin-related protein ( GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.

Author Summary

In canonical clathrin mediated endocytosis (CME) models, the concerted action of ca. 50 proteins mediates the uptake of extracellular components. The key player in this process is clathrin which coats transport intermediates called clathrin coated vesicles (CCV). The intestinal parasite Giardia lamblia has undergone extensive remodeling during colonization of the mammalian duodenum. Here, we report on unique features of this parasite’s endocytic system, consisting of fixed peripheral vacuoles (PV) in close proximity to the exposed plasma membrane (PM), with no discernible CCVs. Using state-of-the-art imaging strategies, we show that the surface of Giardia trophozoites is pock-marked with PM invaginations reaching to the underlying PV membrane. Co-immunoprecipitation and analysis of protein dynamics reveal that, in line with the absence of CCVs, giardial clathrin assemblies have no dynamic behavior. CHC still remains associated to AP2 and dynamin, both conserved dynamic CME components, and to a newly identified putative clathrin light chain. The emerging model calls for giardial clathrin organized into static cores surrounded by dynamic interaction partners, and most likely involved in the regulation of fusion between the PM and the PVs in a “kiss-and-flush”-like mechanism. This suggests that Giardia harbors a conceptually novel function for clathrin in endocytosis, which might be a consequence of host-parasite co-evolution.