Characterisation of Encephalitozoon cuniculi α-actinin

The Encephaliozoon cuniculi is an obligate intracellular microsporidian parasite with a highly reduced genome, yet it contains several key components of an actin cytoskeleton. In this study, we characterise the α-actinin-like protein from the parasite to gain insight into its role in actin organisation. The protein contains three domains typical of α-actinins: an N-terminal actin-binding domain and a C-terminal calmodulin-like domain, separated by a rod domain. Gel filtration analysis demonstrated that the recombinant protein formed stable dimers, consistent with the canonical antiparallel α-actinin structure. Actin co-sedimentation assays and electron microscopy confirmed that the α-actinin-like protein binds and cross-links actin filaments into tight bundles, whereas the isolated actin-binding domain binds but does not cross-link filaments. AlphaFold modelling predicted an overall structural arrangement compatible with an antiparallel dimer. Our results identify the E. cuniculi α-actinin-like protein as a true α-actinin homologue. The presence of actin-binding proteins in E. cuniculi as well as in other microsporidia with very small genomes implies that an actin-based cytoskeleton is important for their survival.