Tale of Two Proteins Sfi1p-like and Centrin: Modeling of Contraction and Relaxation in V. convallaria

Vorticella convallaria, a polymorphic ciliated protozoan tethered to the substrate by an elongated stalk, exhibits a unique contractile system that does not require ATP hydrolysis. The contractile organelles, myonemes and the spasmoneme, are mainly composed of calcium-binding proteins (centrins and spasmins) and are believed to form the 2-5 nm filaments. This calcium-mediated centrin-based cytoskeletal network induces coordinated contraction of the myonemes in the cell body with a concomitant coiling of the spasmoneme of the stalk. A similar fiber network has been described in Paramecium, where the contractile behavior of the infraciliary lattice (a structure equivalent to Vorticella myonemes and spasmoneme) is regulated by the interaction of the two major components: calcium and centrin/centrin-binding (Sfi1-like) protein complex. A centrin-binding (Sfi1-like) protein, a large protein with tightly bound tandemly arranged centrin molecules, constitutes a flexible backbone of the contractile network. In the presence of calcium, the calcium-sensing centrin proteins undergo a conformational change inducing shortening of the filaments and, therefore, supercoiling of the centrin/ centrin-binding (Sfi1-like) protein complex. Immuno-localization studies using anti-human Sfi1 and anti-centrin antibodies in Vorticella, showed that they localize to the myonemes and the spasmoneme. Additionally, the anti-human Sfi1 antibody recognized several oral structures within the cell body including haplo- and polykineties, infundibulum, ciliary wreath, and the cell body-stalk transition zone. Functional studies revealed that microgram quantities of anti-human Sfi1 and anti centrin/spasmin antibodies were sufficient to significantly reduce contraction in glycerol extracted stalk models. We applied a far western technique to search for centrin binding partners. We demonstrated that centrin bound several lower molecular proteins in the range of 20 kDa, suggesting centrin-centerin interactions. Moreover, two higher molecular proteins (over 200 kDa) were detected, result consistent with our hypothesis of the presence of the potential centrin-binding (Sfi1-like) proteins in Vorticella. In modeling the movement mechanism of Vorticella we proposed that this centrin-based cellular motility depends on at least two molecular components, centrins/spasmins and centrin-binding (Sfi1-like) proteins.