Mutant/mutation
The mutant contains a modified/mutated actin-1 gene locus. It contains the wild type actin 1 gene under control of the actin 1 5'-UTR whereas the 3'-UTR is from the dhfs gene. In addition the yfcu::hdfr selection cassette is located in the 3'-UTR region.

This mutant is used as a 'actin-1' recipient line for introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC

Protein (function)
Actin, a cytoskeletal protein, has many diverse functions in eukaryotic cells ranging from roles in cell motility, cell division, vesicle trafficking to functions in cell signaling and regulation of transcription. A critical property of actin is its ability to form filamentous polymers (F-actin), and a plethora of proteins are involved in the highly dynamic regulation of F-actin formation . Actins are highly conserved proteins that often exist in multiple isoforms in the eukaryotic cell and their expression is regulated both spatially and temporally during development. Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself.

The actin monomer has a highly conserved structure that consists of four subdomains and a central nucleotide (adenosine triphosphate [ATP], adenosine diphosphate with inorganic phosphate [ADP + Pi], or adenosine diphosphate alone [ADP]) binding cleft. Actin possesses the ability to self-assemble from monomers (G-actin) to form filaments (F-actin), which in turn can form higher order filamentous structures. Particular regions in the actin subdomains, such as the hydrophobic plug (H-plug) of subdomain 3 and the highly flexible DNAse I-binding loop (D-loop) of subdomain 2, as well as the nucleotide state, have been implicated as major contributors to the formation and stability of filaments

Plasmodium actin only forms short filaments of approximately 100 nm in length, has a noncanonical filament structure that is dynamically unstable, displays slow polymerisation yet rapid depolymerisation rates, and is regulated by a highly reduced set of predicted actin binding proteins (ABPs)(compared to actins of other/higher eukaryotes). Such altered properties are crucial for intracellular parasite growth and efficient parasite motility.

Phenotype
This mutant is used as a 'actin-1' recipient line for introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC

Additional information
Using this 'actin-1 recipient line' a number of parasite mutants have been generated that express mutated forms of actin-1. See the paper for additional mutants expressing mutated forms of actin-1

Analyses of these mutant provided evidence for: 

'modification of most subunit–subunit interaction sites of actin-1 was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient sporozoite motility (and hence mosquito organ penetration). The strong penetration defects could be rescued by over-expression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.'

Other mutants
See the paper for additional mutants expressing mutated forms of actin-1