The Single Dynamin Family Protein in the Primitive Protozoan Giardia lamblia Is Essential for Stage Conversion and Endocytic Transport

Abstract

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominantnegative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominantnegative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

“…We have shown that giActin is associated with the Golgi-like ESVs (Fig. 2D), where the trafficking of CWP is thought to involve clathrin and dynamin (37). To test the role of giActin in CWP trafficking, we encysted actin knockdown cultures.…”

Ancient dynamin segments capture early stages of host–mitochondrial integration

“…There is partial although not conclusive experimental support for this idea. The class A dynamin of the excavate Trypanosoma brucei localizes to mitochondria (25) but appears to be required for mitochondrial fission as well as endocytosis (26); that of the amitochondriate excavate Giardia lamblia colocalizes with clathrin-coated vesicles (27). The class A dynamin of the red alga Cyanidioschyzon merolae has a punctate distribution on the cell surface between mitochondrial division events, suggesting it might also participate in vesicle scission (28,29).…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…In addition, their potential role in endocytosis was demonstrated by the uptake of exogenous ferritin and Lucifer yellow (Bockman and Winborn, 1968;Lanfredi-Rangel et al, 1998). Pulse-chase experiments with horseradish peroxidase and fluorescent dextran showed an early and persistent labeling of the PVs, suggesting that there is no distinction between early and late endocytic vesicles in Giardia, in contrast to what occurs in higher eukaryotes (LanfrediRangel et al, 1998;Gaechter et al, 2008). Analysis of fluid-phase endocytosis demonstrated that there is no lateral exchange of fluid phase markers between individual PVs (Gaechter et al, 2008).…”

“…Pulse-chase experiments with horseradish peroxidase and fluorescent dextran showed an early and persistent labeling of the PVs, suggesting that there is no distinction between early and late endocytic vesicles in Giardia, in contrast to what occurs in higher eukaryotes (LanfrediRangel et al, 1998;Gaechter et al, 2008). Analysis of fluid-phase endocytosis demonstrated that there is no lateral exchange of fluid phase markers between individual PVs (Gaechter et al, 2008). It was observed that, after internalization, some fluid-phase markers translocated rapidly to the ER or to an associated membrane compartment termed the tubulo-vesicular network (TVN) (Abodeely et al, 2009).…”

“…Giardia intestinalis, Giardia duodenalis) is an early branching eukaryote that possesses a reduced membrane network with highly polarized vesicles, denominated peripheral vacuoles (PVs), neighboring the plasma membrane (reviewed by Adam, 2001;Faso and Hehl, 2011;. This is an important zone of interaction between the parasite and its environment (the host intestine in vivo or the culture medium in vitro), and was shown to be the place involved in fluid-phase and receptor-mediated endocytosis (Gaechter et al, 2008;Rivero et al, 2010). Another characteristic of these PVs is that they contain hydrolytic activity resembling the function performed by lysosomes.…”