Protrichocysts: a hybrid defense extrusive organelle bridging mechanical projection and chemical secretion in ciliates

Extrusive organelles (extrusomes) represent a specialized class of dense-core granules in protists that exhibit remarkably complex morphology and secretory mechanisms compared to their metazoan counterparts, reflecting key adaptations in single-celled eukaryote evolution. Protrichocysts, which are among the most intricate projectile extrusomes in ciliates, have thus far been understood primarily in terms of their general morphology. In this study, we employed a comprehensive approach to investigate the protrichocysts of <i>Pseudourostyla cristata</i>, thereby expanding our understanding of these organelles. Through predator-prey interaction experiments and electron microscopy, we demonstrated the ejection of protrichocysts in response to a predator's attack and revealed that their ejection process consists of three main stages: (i) cap dissolution, (ii) dual-phase body expansion, and (iii) controlled shaft exposure. Histochemical analysis indicated that extruded protrichocysts contain acid mucopolysaccharides and microtubules. SDS-PAGE and HPLC-MS/MS analyses identified the major protein bands and the presence of glycoproteins, phosphoproteins, and potential defense-related proteins. Single-cell transcriptome analysis suggested the involvement of conserved carboxypeptidase-mediated extrusome biogenesis. These findings establish protrichocysts as multifunctional organelles that employ a unique hybrid defense strategy combining mechanical projection with chemical secretion. Their distinctive microtubular cap architecture and specialized matrix composition further suggest potential roles in intercellular communication. Additionally, this research improves our understanding of protist extrusome biology and provides new insights into the evolution of eukaryotic secretory systems.