Abstract
ABSTRACTEncystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-RacCA) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis.
Highlights
Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM)
We found that HA-Rac was associated with the perimeter of cyst wall protein 1 (CWP1)-positive vesicles known as encystation-specific vesicles (ESVs) (Fig. 1B)
We conclude that GlRac has a critical regulatory role in encystation, based on four observations: (i) GlRac associates with the endoplasmic reticulum (ER) and ESVs; (ii) GlRac is temporally upregulated during encystation; (iii) constitutive GlRac signaling causes ER swelling and reduces cellular CWP1 levels; and (iv) constitutive GlRac signaling reduces encystation rates as well as disrupts cyst wall integrity
Summary
Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). We examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. Studies of G. lamblia, which is itself placed in a critical, albeit controversial, deep-branching position on the tree of life [33,34,35], may provide insight into the ancestral function of Rho family GTPases
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