An Unconventional Form of Actin in Protozoan Hemoflagellate, Leishmania

Abstract

Leishmania actin was cloned, overexpressed in baculovirusinsect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg 2؉ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg 2؉ alone, (ii) it polymerized in a restricted range of pH 7.0 -8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg 2؉ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.

ADF/cofilin-driven actin dynamics in early events ofLeishmaniacell division

“…That actin in trypanosomatids is involved in intracellular vesicular trafficking and flagellar-pocket organization is further supported by an earlier study which showed that loss of actin prevents endocytosis and results in enlargement of the flagellar pocket in the bloodstream form of T. brucei (Garcia-Salcedo et al, 2004). However, it is important to mention here that the Leishmania F-actin by itself displays fast dynamics compared with conventional actins (Kapoor et al, 2008), and therefore some monomeric actin is always available to drive the actin dynamics, although at a reduced pace, even in the absence of any actin-dynamics-regulating protein. It might therefore be visualized that deletion of the Leishmania COF gene has only slowed down, but not abolished, the dynamics of filamentous actin, which has resulted only in the slowing down of the actin-based processes.…”

“…In higher organisms, ADF/cofilin is essential for depolymerization of the actin contractile ring that forms between two daughter cells during cell division (Abe et al, 1996). However, in the case of trypanosomatid parasites, cytokinesis is primarily a microtubule-mediated process, and despite the presence of actin and several actin-binding proteins (Sahasrabuddhe et al, 2004;Nayak et al, 2005;Kapoor et al, 2008;Tammana et al, 2008;Katta et al, 2009), their role in these parasites was largely unknown. Our recent studies (Tammana et al, 2008;Sahasrabuddhe et al, 2009) show that the actin-binding proteins Cof and CRN12 have essential roles in flagellar assembly and microtubule remodeling, respectively.…”

“…Detailed characterization of Leishmania ADF/cofilin (Cof) revealed that it readily binds both the monomeric and filamentous (Kapoor et al, 2008) forms of Leishmania actin and also depolymerizes filamentous actin into monomers (Tammana et al, 2008). Furthermore, it displays high nucleotide exchange but weak actin-filament-severing activities (Tammana et al, 2008).…”

Overexpression of S4D Mutant of Leishmania donovani ADF/Cofilin Impairs Flagellum Assembly by Affecting Actin Dynamics

“…By virtue of these activities, ADF/cofilins play a key role in regulating the actin dynamics and associated functions in eukaryotes (7). Functions of the actin cytoskeleton have been considered important not only in higher eukaryotes but also in several parasites that cause lifethreatening human diseases, such as Plasmodium (5,49), Acanthamoeba (10,23), Trypanosoma (13,21), Leishmania (30,47), and others.…”

AncientLeishmaniacoronin (CRN12) is involved in microtubule remodeling during cytokinesis

“…We also reported that Leishmania CRN12 associates with filamentous actin in Leishmania promastigotes (Nayak et al, 2005;Kapoor et al, 2008), and is retained in the flagellar pocket region and in a few cells at the distal tip (Nayak et al, 2005). We have now further explored the intracellular distribution of Leishmania CRN12 in both the resting and dividing Leishmania cells.…”