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

Abstract

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. cytoskeleton evolution | actin protofilaments | MreB | endocytosis | Rac

Interplay between Two Bacterial Actin Homologs, MamK and MamK-Like, Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

“…In most eukaryotes, actin is highly conserved and its function and dynamics are impacted by a standard set of actin binding proteins. Interestingly, the intestinal parasite Giardia intestinalis contains one of the most divergent eukaryotic actins and also lacks many of the canonical actin binding proteins (44). This fascinating "exception to the rule" supports a hypothesis that preservation of essential interactions between actin and its conserved regulatory partners is a key evolutionary constraint that limits the protein's sequence divergence among eukaryotes.…”

Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

“…That giActin performs similar functions to actin in other eukaryotes suggests these processes were already associated with actin at the time Giardia split from the other eukaryotes (13). We have also shown that giRac, the sole Rho family GTPase in Giardia, regulates actin despite the absence of all actin-binding proteins known to link G-protein signaling to the actin cytoskeleton (Arp2/3, formin, wave, myosin, and cofilin) (13). Therefore, Giardia must contain a novel set of actin-interacting proteins comprised of ancient yet undiscovered and/or Giardia-specific actin regulators.…”

“…Their absence may indicate a split from the last eukaryotic common ancestor before the canonical set of actin-binding proteins was established. Alternatively, Giardia may have evolved a novel set of actin-interacting proteins that allowed for the gradual loss of the canonical set of actin-binding proteins (11,13). Our previous work has shown that despite the lack of canonical actinbinding proteins, Giardia actin (giActin) is required for conserved cellular functions, including membrane trafficking, cytokinesis, polarity, and control of cellular morphology (13).…”

“…After 1.5 h of binding, the beads were washed four times with wash buffer (25 mM Tris 7.5, 150 mM NaCl, 0.25 mM CaCl 2 , 0.25 mM ATP, 5% glycerol, 0.05% Tween 20) and then boiled in 50 l of sample buffer. Western blotting was performed as described previously (13). Rabbit anti-giActin polyclonal (13) and anti-HA mouse monoclonal HA7 antibody (Sigma-Aldrich) were both used at 1:3,000.…”

The Giardia cell cycle progresses independently of the Anaphase Promoting Complex

“…At 24 hours after transfection cells were harvested and either lysed and analyzed by western blotting or fixed for immunofluorescence. For western blots, cells were harvested, lysed in Laemmli SDS sample buffer, resolved on a 10% acrylamide gel that was then transferred to a non-fluorescent PVDF membrane and probed using anti-HA (HA-7, Sigma, 1:3000), anti-MPM2 (MPM2, Millipore, 1:1000) or anti-actin (Paredez et al, 2011) (1:3000), followed by fluorescent secondary antibody (IRDye680-linked anti-mouse and IRDye800-linked anti-rabbit, Li-Cor). Signals were detected and quantified using the Odissey infrared imager.…”