Toxoplasma gondii Profilin Acts Primarily To Sequester G-Actin While Formins Efficiently Nucleate Actin Filament Formation in Vitro

Abstract

Apicomplexan parasites employ gliding motility that depends on the polymerization of parasite actin filaments for host cell entry. Despite this requirement, parasite actin remains almost entirely unpolymerized at steady state; formation of filaments required for motility relies on a small repertoire of actin-binding proteins. Previous studies have shown that apicomplexan formins and profilin exhibit canonical functions on heterologous actins from higher eukaryotes; however, their biochemical properties on parasite actins are unknown. We therefore analyzed the impact of T. gondii profilin (TgPRF) and FH1-FH2 domains of two formin isoforms in T. gondii (TgFRM1 and TgFRM2) on the polymerization of T. gondii actin (TgACTI). Our findings based on in vitro assays demonstrate that TgFRM1-FH1-FH2 and TgFRM2-FH1-FH2 dramatically enhanced TgACTI polymerization in the absence of profilin, making them the sole protein factors known to initiate polymerization of this normally unstable actin. In addition, T. gondii formin domains were shown to both initiate polymerization and induce bundling of TgACTI filaments; however, they did not rely on TgPRF for these activities. In contrast, TgPRF sequestered TgACTI monomers, thus inhibiting polymerization even in the presence of formins. Collectively, these findings provide insight into the unusual control mechanisms of actin dynamics within the parasite.

Gliding motility powers invasion and egress in Apicomplexa

“…Second, the apicomplexans exhibit a smaller repertoire of actin-binding proteins (ABPs) than many lower eukaryotes, and they lack one of the major actin-nucleating machineries, the actinrelated protein 2/3 (ARP2/3) complex 32 . Last, two ABPs -actin-depolymerizing factor (ADF) and profilinunexpectedly contribute to the high monomer content by sequestering free G-actin 33,34 (FIG. 4b).…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…In P. falciparum, PfFRM1 was reported to localize at the apical pole of free merozoites and to follow the tight junction where the glideosome is predicted to power movement. The FH2 domains of these two formins enhance actin polymerization in the presence of Toxoplasma actin in vitro (Skillman et al, 2012). In those same assays, the biochemical properties of TgPRF indicate that the protein sequestrates actin monomers and does not contribute to polymerization (Skillman et al, 2012).…”

“…The FH2 domains of these two formins enhance actin polymerization in the presence of Toxoplasma actin in vitro (Skillman et al, 2012). In those same assays, the biochemical properties of TgPRF indicate that the protein sequestrates actin monomers and does not contribute to polymerization (Skillman et al, 2012). TgADF is another highly potent factor that leads to actin depolymerization, and deletion of the gene results in the accumulation of actin filaments in T. gondii (Mehta and Sibley, 2011).…”

Un complexe moléculaire unique à l’origine de la motilité et de l’invasion des Apicomplexes

“…Plusieurs protéines liant l'actine et conservées à travers le phylum ont d'ailleurs récemment été décrites, et leur rôle s'est révélé critique pour la motilité et l'invasion du toxoplasme. Les formines TgFRM1 (Toxoplasma formin 1) et TgFRM2 sont impliquées dans la polymérisation des filaments d'actine [16,17], alors que la cofiline TgADF (Toxoplasma acting depolymerizing factor) joue un rôle dans leur dépolymérisation [18,19] et que la profiline TgPRF (Toxoplasma profilin) séquestre les monomères [17,20] (Figure 2B). Normalement, formine et profiline fonctionnent de façon coopérative afin de stimuler la formation du filament d'actine de part et d'autre du domaine FH2 (formin homology 2 domain) de la formine ( Figure 2B).…”

“…De plus, elles fonctionnent de manière coordonnée au niveau de la pellicule pour générer de courts filaments au moment même de la motilité [16]. Par conséquent, en l'absence de complexe Arp2/3, TgFRM1 et TgFRM2 apparaissent comme les principaux facteurs de nucléation des filaments d'actine chez les Apicomplexes, alors que la profiline contribue à maintenir l'actine sous une forme globulaire [17]. Chez Plasmodium, une des formines (PfFRM1) est localisée au niveau du pôle apical du parasite et forme un anneau très précis qui suit la jonction mobile au cours de l'entrée du mérozoïte dans le globule rouge [21].…”