Membrane traffic and Ca²⁺ signals in ciliates

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca²⁺ -regulating proteins, including Ca²⁺ -channels, Ca²⁺ -pumps, Ca²⁺ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca²⁺ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H⁺ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca²⁺ -influx channels, locally enriched intracellular InsP₃ -type (InsP₃ R, mainly in osmoregulatory system) and ryanodine receptor-like Ca²⁺ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca²⁺ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca²⁺ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca²⁺ -influx, that is, a mechanism called "store-operated Ca²⁺ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca²⁺ -concentration, [Ca²⁺ ]_i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca²⁺ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.