Twinfilin is an evolutionarily conserved actin-binding protein, which regulates actin-dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.

Leishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmania actin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum. Reorganization of actin cytoskeleton is central to several fundamental processes in eukaryotes, including cell division, cell shape regulation and, transmission of extracellular stimuli toward the cell interior. Such diverse functions of actin cytoskeleton have been attributed to the dynamic character of actin, which requires high turnover of actin monomers in its filamentous meshwork by a treadmilling process (11). This process is greatly facilitated by the actin-depolymerizing protein (ADF)/cofilin family of actin binding proteins (40). These proteins generally have three distinct biochemical activities, viz., F-actin depolymerization, actin filament severing, and nucleotide exchange (12). 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.Leishmania spp. constitute a group of medically important protozoan parasites that are responsible for a vast array of devastating human diseases, including kala-azar (visceral leishmaniasis). These organisms exist in two morphobiological forms, amastigotes (inside the human host) and promastigotes (in the insect vector), which undergo extensive cytoskeletal rearrangement during their transformation from one form to the other (25). The promastigote form possesses a single highly motile protruding flagellum, which drives the cell to move forward, whereas the rudimentary flagellum in amastigotes has been considered important to establish host-parasite interactions (22). Further, a direct involvement of the promastigote flagellum has been demonstrated in sandfly infection (16). Apart from being ...

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