| 2005 |
ACT1 (actin I) in P. falciparum merozoites carries an N-terminal acetylation (acetyl-Gly-Glu-actin) as its only post-translational modification; methylation of histidine 73, common in canonical actins, is absent. Approximately half of extracted monomeric ACT1 polymerises into filaments, but these filaments are very short (~100 nm average length by EM), too short to be resolved by conventional fluorescence microscopy. In an in vitro motility assay, ACT1 filaments moved over myosin class II at the same velocity as rabbit skeletal actin. |
Mass spectrometry (PTM identification), sedimentation at 500,000 g (F-actin quantification), electron microscopy (filament length), in vitro motility assay |
Journal of molecular biology |
High |
15876372
|
| 2005 |
Recombinant ACT1 produced in yeast polymerises inefficiently unless both gelsolin and phalloidin are added, yielding fragmented polymers. ACT1 binds DNaseI approximately 200-fold more weakly than bovine non-muscle actin, indicating unusual monomer properties that likely underlie the short, unstable filaments observed in apicomplexan parasites. |
Recombinant protein expression in yeast, in vitro polymerisation assay, fluorescence microscopy, DNaseI binding assay |
FEBS letters |
Medium |
15670824
|
| 2010 |
ACT1 (Pf-actin I) stabilised by jasplakinolide forms long filaments in vitro. Atomic force microscopy shows the helical pitch of the ACT1 double-stranded filament is ~10% larger than that of rabbit skeletal actin, corresponding to a larger rotational angle between subunits, consistent with weaker inter- and intra-strand contacts that explain inherent filament instability. Hybrid filaments (ACT1 seeds elongated with rabbit skeletal actin) are stable enough to move over myosin class II motors. |
Atomic force microscopy, negative-stain electron microscopy, fluorescence microscopy, gliding filament assay |
The Journal of biological chemistry |
High |
20826799
|
| 2011 |
In P. berghei, ACT1 (actin I) localises dually to the cytoplasm and nucleus in male gametocytes before activation; after activation it is restricted to the cytoplasm. In actin II knock-out parasites, this re-localisation is abolished and ACT1 remains in both compartments, placing ACT1 re-distribution downstream of actin II function during male gametogenesis. |
Genetic disruption of actin II in P. berghei, immunofluorescence microscopy of ACT1 localisation before and after gametocyte activation |
Cellular microbiology |
Medium |
21790945
|
| 2011 |
A conformation-specific peptide antibody (Ab-actinI-I) raised against the divergent N-terminus of ACT1 detects rod-like F-actin structures (~600 nm) in motile P. berghei ookinetes in vivo; these structures are sensitive to inhibitors of actin polymerisation, demonstrating that ACT1 forms filamentous structures in ookinetes. |
Peptide antibody generation, immunofluorescence microscopy, pharmacological inhibition of actin polymerisation |
Molecular and biochemical parasitology |
Medium |
22101204
|
| 2011 |
Pharmacologically induced F-actin polymerisation of ACT1, which is restricted to the nuclear periphery in P. falciparum, repositions var intron-carrying episomes and var genes from repressive to transcriptionally active perinuclear compartments, disrupting mutually exclusive var gene expression. This places ACT1 polymerisation as a direct mechanistic driver of var gene repositioning and antigenic variation. |
Pharmacological induction of F-actin, fluorescence in situ hybridisation (FISH) for gene positioning, reporter assays for var gene expression, identification of actin-recruiting nuclear element |
Cell host & microbe |
Medium |
22100161
|
| 2014 |
Crystal structures of ACT1 monomers, combined with cryo-EM of filaments, reveal that ACT1 has a unique filament structure distinct from canonical F-actin. Both ACT1 and actin II hydrolyse ATP more efficiently than α-actin, and unlike any other actin, both isoforms rapidly form short oligomers induced by ADP. Inserting the canonical D-loop into ACT1 leads to long filament formation in vitro and, in vivo, restores male gametogenesis in P. berghei parasites lacking actin II, demonstrating that the D-loop is the structural element governing filament stability and length. |
X-ray crystallography, cryo-EM, in vitro polymerisation assay, ATP hydrolysis assay, D-loop chimera mutagenesis, genetic complementation in P. berghei |
PLoS pathogens |
High |
24743229
|
| 2017 |
Near-atomic cryo-EM structure of jasplakinolide-stabilised ACT1 filaments reveals small but critical differences at inter- and intra-strand contact sites compared to mammalian F-actin, explaining the inherent instability of ACT1 filaments. Jasplakinolide binds at regular intervals inside the filament, contacting three adjacent actin subunits and reinforcing stability via hydrophobic interactions. |
Electron cryomicroscopy (near-atomic resolution), jasplakinolide-stabilised filament reconstitution |
Proceedings of the National Academy of Sciences of the United States of America |
High |
28923924
|
| 2017 |
Conditional Cre-lox depletion of ACT1 in P. falciparum blood stages demonstrates that ACT1 is required for: (1) segregation of the apicoplast, (2) efficient daughter cell separation during cytokinesis, and (3) host cell invasion after junction formation. Egress from the host cell is not actin-dependent. Parasites lacking ACT1 can secrete micronemes, attach to erythrocytes, and form a tight junction, but cannot complete invasion. |
Conditional Cre-lox genetic depletion, fluorescence and electron microscopy, invasion assays, microneme secretion assay |
BMC biology |
High |
28810863
|
| 2017 |
ACT1 polymerises via the classical nucleation-elongation pathway with kinetics similar to canonical actins, not via the isodesmic mechanism proposed for Toxoplasma gondii actin. At steady state, ACT1 is present in approximately equal amounts of short filaments and dimers with a small proportion of monomers; the apparent critical concentration is ~0.1 µM. A high fragmentation rate governed by weak lateral contacts is the primary determinant of short filament length. |
Quantitative in vitro polymerisation assays with modified fluorescent labelling, kinetic analysis |
Scientific reports |
Medium |
28939886
|
| 2017 |
Reconstitution of the glideosome core with recombinant PfMyoA (expressed via baculovirus/Sf9) and ACT1 shows that PfMyoA moves ACT1 filaments at the same speed as skeletal muscle actin filaments. The duty ratio of PfMyoA suggests that as few as nine motors can power actin movement at maximal speed, a property potentially necessitated by the dynamic (short) nature of ACT1 filaments in the parasite. |
Baculovirus/Sf9 recombinant protein expression, in vitro motility assay, actin-activated ATPase assay |
The Journal of biological chemistry |
Medium |
28978649
|
| 2017 |
In P. berghei gametocytes (non-motile), ACT1 is present in discrete foci of <130 nm (resolved by 3D-structured illumination microscopy), consistent with short cross-linked filaments anchored to cytoskeletal structures. A portion of ACT1 is soluble, a portion is in a stabilised form requiring SDS for solubilisation, and an additional population pellets at 100,000 × g consistent with F-actin. |
3D-structured illumination microscopy, differential solubilisation/fractionation, immunofluorescence with F-actin-specific antibody |
Experimental parasitology |
Medium |
28803903
|
| 2019 |
High-resolution crystallography shows that Mg2+ binding causes slight flattening of the ACT1 monomer (closer in conformation to F-actin), and subsequent phosphate release results in a more twisted conformation. A coordinated K+ ion in the active site departs with phosphate; the A-loop (Arg178/Asp180) acts as a conformational switch between stable and unstable filament states by alternating interactions with Lys270 or His74. His74 in ACT1 is unmethylated (unlike canonical His73-methyl actin), contributing to the fragmentation propensity. |
High-resolution X-ray crystallography, active-site structural analysis with metal ions and nucleotide states |
PLoS biology |
High |
31199804
|
| 2019 |
TIRF microscopy of individual unstabilised ACT1 filaments shows that ACT1 can form filaments up to 30 µm but with a critical concentration of ~4 µM (an order of magnitude higher than skeletal actin). Protomers dissemble from both barbed and pointed ends at similar fast rates of 10–15 subunits/s, and rapid treadmilling occurs near the critical concentration. ADP-ACT1 filaments are highly unstable, dissolving completely in <40 min; ADP-Pi or BeFX suppresses end dynamics. A chimeric ACT1 bearing the mammalian D-loop forms more stable filaments. |
Total internal reflection fluorescence (TIRF) microscopy of individual filaments, critical concentration measurement, kinetic analysis of barbed/pointed end dynamics, chimeric D-loop mutagenesis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
31548388
|
| 2021 |
Cryo-EM structure of the P. falciparum ACT1–PfMyoA actomyosin complex at the rigor (end-of-powerstroke) state reveals the detailed ACT1:PfMyoA interface. The footprint of PfMyoA on filamentous ACT1 is conserved with higher eukaryote actomyosin complexes despite important variability in the contact residues. The interface comprises a conserved core common to all actomyosin complexes plus an ancillary region defining the spatial positioning of the motor on ACT1 filaments. |
Electron cryomicroscopy (atomic resolution), structure determination of rigor-state actomyosin complex |
Nature communications |
High |
33767187
|
| 2022 |
High-resolution cryo-EM structure of the full glideosome motor complex reveals the ACT1 filament (PfAct1) at improved resolution complexed with PfMyoA and its two light chains (PfELC and PfMTIP), providing atomic-detail view of the ACT1:PfMyoA interface and the light-chain binding region at lower resolution. A bare ACT1 filament structure is also reported at improved resolution. |
Electron cryomicroscopy (high resolution), structural determination of actomyosin and bare filament |
PLoS pathogens |
High |
35377914
|