The conserved ancestral signaling pathway from cilium to nucleus.

Many signaling molecules are localized to both the primary cilium and nucleus. Localization of specific transmembrane receptors and their signaling scaffold molecules in the cilium is necessary for correct physiological function. After a specific signaling event, signaling molecules leave the cilium, usually in the form of an endocytic vesicle scaffold, and move to the nucleus, where they dissociate from the scaffold and enter the nucleus to affect gene expression. This ancient pathway probably arose very early in eukaryotic evolution as the nucleus and cilium co-evolved. Because there are similarities in molecular composition of the nuclear and ciliary pores the entry and exit of proteins in both organelles rely on similar mechanisms. In this Hypothesis, we propose that the pathway is a dynamic universal cilia-based signaling pathway with some variations from protists to man. Everywhere the cilium functions as an important organelle for molecular storage of certain key receptors and selection and concentration of their associated signaling molecules that move from cilium to nucleus. This could also have important implications for human diseases such as Huntington disease.

Functional interaction between autophagy and ciliogenesis.

Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signalling motivated us to explore the possible functional interactions between this signalling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signalling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. Whereas abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signalling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie some common ciliopathies.

RNAi knockdown of parafusin inhibits the secretory pathway.

Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.

Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca(2+)-regulated exocytosis.

The Paramecium tetraurelia protein parafusin (PFUS) and the Toxoplasma gondii protein parafusin-related protein 1 (PRP1) both have two covalent modifications (phosphorylation and phosphoglucosylation) and both are members of the phosphoglucomutase superfamily, associating with secretory vesicle scaffolds in their respective cells. This study tests the hypothesis that PRP1 is a functional ortholog of PFUS, functioning identically in Ca(2+)-regulated exocytosis. Electroporation of fluorescently labeled recombinant His-PRP1 into live Paramecium cells resulted in its localization to docked, dense-core secretory vesicles (DCSVs) in a pattern identical to endogenous PFUS. In tam8 mutants, defective in transport of DCSVs, the fluorescently labeled protein was restricted to the un-transported DCSVs. Specificity of PRP1 localization was demonstrated by electroporating labeled actin or pyruvate kinase, which both failed to localize to either docked or undocked vesicles. In wild-type Paramecium, electroporated His-PRP1 dissociated from DCSVs upon exocytosis, and re-associated as new organelles formed. Mutagenized His-PRP1 species (S146A or S146E) cannot be phosphorylated by P2 calcium-dependent kinase in vitro. Upon electroporation, these molecules remained cytoplasmic and un-associated with DCSVs, while mutation of another PRP1 serine residue (S560A) did neither affect the localization to the DCSVs nor the phosphorylation pattern. Therefore, in this heterologous system, localization, transport and dissociation/re-association of PRP1 substituted for PFUS, supporting the conclusion that the proteins are functional orthologs. The assay also identified a strategic residue S146 within the PFUS ortholog (S138 in PFUS by extrapolation) required for post-translational modification, DCSV scaffold association and for exocytosis.

Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii.

The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.