Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada

Abstract

Background Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. Results Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. Conclusions Taken together, this provides the first comprehensive nanometric view of Giardia’s endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

CHC22 clathrin functions in the early secretory pathway by two-site interaction with SNX5 and p115

“…In so far as SNX5 interacts with the TxD, it may provide the trimer-stabilizing function imparted by CLC to CHC17 (Ybe et al , 2007). Indeed, recent phylogenetic studies identifying CLCs of highly divergent sequences in protist eukaryotes (Santos et al , 2022) indicate that CLC equivalents are predicted to interact with the TxD of CHC17 equivalents, and that this may be the essential molecular requirement for a CHC-associated subunit. As SNX5/6 interacts principally with the TxD of CHC22 rather than spanning the proximal leg of the heavy chain to the knee where CLC affects CHC17 assembly dynamics (Redlingshöfer & Brodsky, 2021), one possibility is that SNX5/6 binding influences the CHC22 uncoating dynamics.…”

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

“…The clathrin heavy chain is retained in the vast majority of eukaryotes, where it performs a well‐observed role, serving as one of the several interaction hubs operating within the endosomal system (Schmid & McMahon, 2007 ). By contrast, the regulatory light chain is less frequently detected and at least in the diplomonad flagellates (including G. lamblia ), appears to have been functionally replaced by an analogous protein (Santos et al ., 2022 ). Furthermore, only few of the conventional clathrin protein partners, i.e.…”

ImagingGiardia intestinaliscellular organisation using expansion microscopy revealed atypical centrin localisation

“…by light and electron microscopy in Nohýnková et al (2006) and by ExSIM in Halpern et al (2017). The trophozoite cell contains two nuclei (Tůmová et al 2016) and many parasitic life-adapted organelles, such as a mesh of peripheral vacuoles (Santos et al 2022) or highly reduced mitochondria (Tůmová et al 2021). The ExM pipeline presented here enables imaging of the structural context of the whole Giardia cell, yielding high resolution information about specific protein localisation, such as that of centrin.…”