Fig. 6: Schematic representation of a proposed intracellular trafficking of calcifying medium in the calicoblastic cells of S.

pistillata. (1) Ion and molecules from seawater and small fluorescent probes (red) gain access to the extracellular calcifying medium, sandwiched between the skeleton and the apical pole of calicoblastic cells, through the paracellular pathway^26,52. (2) the extracellular calcifying medium enters the calicoblastic cells via macropinocytosis²⁶. After being endocytosed at the apical membrane, macropinosomes containing calcifying medium undergo intracellular traffic and sorting as endosomes (proposed in current study). (3) A fraction of the endosomes transiently acquires SpiRab11 (magenta), which promotes their recycling towards the plasma membrane (proposed in current study). (4) The skeletal organic matrix proteins (SOM) (orange) synthetized in the endoplasmic reticulum and the Golgi complex is delivered in the extracellular CM through the secretory pathway and partly endocytosed back by the calicoblastic cells, along with CM, through macropinocytosis (proposed in current study). A fusion of recycling endosomes with secretory vesicles budding from the trans-Golgi network (TGN) may exist but we have not observed it. Note that a fraction of the endocytosed CM might undergo fast recycling through the Rab4-dependent pathway, or be directed towards the late endosomal/lysosomal compartments (proposed in current study).