Secretory Mechanisms in Paramecium

Abstract

A Paramecium cell possesses two widely different types of secretory activity. One is the release from dense core secretory organelles, the trichocysts, serving for predator defense by the most rapid known synchronous process: single events lasting <1 ms and <80 ms for the whole population of stimulated cells. The second type is the periodic release of water and excess of ions, notably Ca²⁺, by the contractile vacuole complex which thus serves for osmoregulation and ionic balance. Trichocyst secretion encompasses organelle docking at regularly placed sites at the cell membrane, with assembly of SNARE proteins and an undefined Ca²⁺ sensor. Upon stimulation and increase in [Ca²⁺], membrane fusion and release of contents take place with a speed and synchrony unsurpassed by other dense core vesicle systems. Also exocytosis–endocytosis coupling is relatively fast (<270 ms), so that the entire process takes 350 ms. The apparent time constants are, thus, τ _exo = 57 ms and τ _endo = 126 ms; the decay of the cortical Ca²⁺ signal proceeds with τ = 8 s. Contact with exogenous Ca²⁺ after exocytotic pore formation triggers automatically explosive decondensation (several-fold elongation of the spindle-shaped trichocyst contents). The two contractile vacuoles are permanently docked at specific sites of the cell membrane. Their membrane fuses with the cell membrane, also at epigenetically predetermined sites, periodically every ~10 s, when luminal osmotic pressure has sufficiently increased, probably by mechanosensitive channels enriched by the scaffolding protein, stomatin. Both types of secretory activity depend on local Ca²⁺ increase. In the case of trichocysts, Ca²⁺ for membrane fusion is provided by store-operated Ca²⁺ entry (SOCE) in synchrony with the release of Ca²⁺ from alveolar sacs, the cortical Ca²⁺ stores, via ryanodine receptor-like Ca²⁺-release channels. Remarkably, contractile vacuole complexes also use SNAREs and they are also surrounded by Ca²⁺ stores, but here the mechanism of [Ca²⁺]_i increase for membrane fusion remains to be determined.

Key References: See Main List for Reference Details

• Bright et al. (2010) The authors produced GFP-labeled Rab-type GTPases in vivo in T. thermophila cells to follow the pathways of organelle trafficking. Data are well comparable with metazoans.

• Guerrier et al. (2017) This paper tries to sound out the compromises between conservation and change in protein molecules and their function during evolution.

• Hardt and Plattner (2000) A combination of innovative technologies, including quenched-flow/fast freezing combined with electron microscopy (see Knoll et al. 1991, below), has been applied to tackle the problem of rapid intracellular Ca²⁺ shifts during exocytosis stimulation in Paramecium and to “fix” and localize Ca²⁺ by X-ray microanalysis in submicroscopic domains without artificial redistribution.

• Kissmehl et al. (2002) This was one of the first approaches to establish the crucial role of SNARE proteins for membrane trafficking and exocytosis in a unicellular organism (P. tetraurelia). Later on, this was followed by identification, localization, and functional analysis of SNAREs (see papers by Schilde and Kissmehl).

• Knoll et al. (1991) The goal was to analyze in P. tetraurelia rapid ultrastructural changes of exocytosis sites during sub-second times of stimulation. One of the problems was to avoid side effects by mechanical strain.

• Ladenburger and Plattner (2011) Ladenburger and coauthors identified for the first time in a protozoan the most important intracellular Ca²⁺-release channels, such as InsP₃ and ryanodine receptors (RyR-LPs), followed by functional studies (e.g., posttranscriptional downregulation and effects on Ca²⁺ signaling) as well as light and electron microscopic localization.

• Plattner (2014a) This review summarizes the “calcium adventure” with Paramecium.

• Plattner (2017a) This review summarizes the broad spectrum of signaling mechanisms in ciliated protozoa.

• Plattner and Verkhratsky (2018) discuss the maintenance of important proteins and regulatory mechanisms from protozoa to mammalian brain.

• Stelly et al. (1991) Stelly and associates were the first to prove by biochemical methods that alveolar sacs are cortical Ca²⁺ stores in ciliates.

• Wassmer et al. (2009) The numerous subunits of the H⁺-ATPase have been identified and differentially localized to trafficking vesicles in P. tetraurelia. Most surprising was the bewildering number of 17 a-subunits (only 4 in man) that reversibly link the catalytic V1 part with the membrane-integrated V0 basepiece. Hypothetically this could adjust pump kinetics to local requirements.