Reconstitution of the calcium‐triggered contractile apparatus of ciliated protozoa

Protists, notably ciliated protozoa, exhibit a novel form of contraction that is rapidly triggered by calcium ions but does not require ATP hydrolysis. Ultrafast forms of calcium-triggered contraction had previously been described for the Vorticella stalk and the Spirostomum cell body. In these ciliates, cell structures (stalk or body) contract within a few milliseconds. Spasmin, an EF-hand calcium binding contractile protein was identified as the key component of the Vorticella spasmoneme, the contractile organelle present within its stalk. In heterotrich ciliates, such as Stentor and Spirostomum, contraction is mediated by branching, spindle-shaped structures called myonemes, which contain centrin-like proteins. Bioinformatics analysis of published and recently obtained data revealed the existence of several distinct groups of EF-hand calcium-binding contractile proteins in ciliated protozoa. Synthetic genes encoding these calcium-binding proteins, as well as their putative scaffold proteins, have been engineered for expression in bacteria. In vitro reconstitutions of these contractile systems were then attempted using purified proteins. Tcb2 protein from Tetrahymena thermophila was identified as a major component of contractile gels formed when calcium ions were added to a low ionic strength alkaline extract of the Tetrahymena membrane-associated cytoskeleton. Purified Tcb2 protein showed calcium-dependent reversible assembly in microscopic, spectroscopic, and pelleting assays. Tcb2 has also been shown to interact with and drive the collapse of gels formed with either of two other alpha-helical coiled-coil cytoskeletal proteins, Epc1 and Fen1. Through proteomic analysis of Spirostomum cytoskeletal preparations, a family of EF-hand proteins closely related to the calcium-binding protein centrin have been identified. In addition, a putative centrin-binding Sfi1-like scaffold protein has been identified in Spirostomum. Proteins encoding putative components of the Spirostomum ambiguum myoneme contractile systems have been expressed in bacteria and purified. Solutions of these Spirostomum proteins responded to calcium ions in a similar way to that seen for Tetrahymena contractile proteins. Our immediate goal is to engineer Spirostomum contractile proteins to fully reconstitute in vitro the ultrafast myoneme contraction taking place in living Spirostomumcells. These studies should yield insight into the structure, mechanism and regulation of the calcium-triggered contractile systems present in protistan cells. As a parallel goal, we are working to engineer optically controlled contractile elements built from these proteins, and introduce them into metazoan animal cells and synthetic cells, so that we can manipulate their behaviors through patterned light activation.