Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

Abstract

The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure).

Principles of Intracellular Signaling in Ciliated Protozoa—A Brief Outline

“…This may serve not only for fine tuning of cytosolic Ca 2 + but also to drive the extensive membrane flL<;ion and fission processes within the organelle during systole/diastole cycles (Plattner 2015b). Pore for periodic contents release by exocytosis: with specific SNAREs and CRCs at the pore periodic signal for vacuole contents release: mechanosensitive channels (suggested by occurrence ofstomatin [Reuter et al 2013] and in agreement with other systems [Plattner 2015b]), in conjunction with pore-specific SNAREs and CRCs…”

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics

“…Irregular, stochastic Ca 2+ puffs are registered with fluorochromes along radial arms; they are attributed to constitutive activity of Pt CRC‐II/InsP 3 R channels (Ladenburger et al, ). The presence of stomatin, which in Paramecium tetraurelia is found from the peripheral parts of the radial arms up to the pore (Reuter, Stuermer & Plattner, ), suggests the occurrence of mechanosensitive channels since both molecules are always combined with each other in other systems (see Section VIII.2). Therefore, increasing pressure may activate such channels, possibly in conjunction with some of the other CRCs.…”

“…The occurrence of intracellular mechanosensitive channels in Paramecium tetraurelia cells has been postulated on the basis of the presence of stomatin in food vacuoles and in the contractile vacuole complex up to its pore region (Reuter et al, ). This scaffolding protein is normally associated with microdomains and, on a molecular level, with mechanosensitive channels for whose activity it is obligatory (Lapatsina et al, ).…”

Evolutionary Cell Biology of Proteins from Protists to Humans and Plants

“…When we analyzed the possible occurrence of members of the SPFH superfamily in ciliates, we realized that they may be closer to stomatin in P. tetraurelia , but closer to reggie/flotllin in T. pyriformis (Reuter et al. ). Occurrence of flotillin 1 (reggie 2) and of flotillin 2 (reggie 1) is reported from database search in choanoflagellates (Burkhardt ; Burkhardt et al.…”