A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N–C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM inChlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1^−/− mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1^−/− multiciliated ependymal cells and human PCM1^−/− retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1^−/− RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1^−/− ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.

Ciliary axonemes and basal bodies were present in the last eukaryotic common ancestor and play crucial roles in sensing and responding to environmental cues. Peptidergic signaling, generally considered a metazoan innovation, is essential for organismal development and homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) is crucial for the last step of bioactive peptide biosynthesis. However, identification of a complete PAM-like gene in green algal genomes suggests ancient evolutionary roots for bioactive peptide signaling. We demonstrate that the Chlamydomonas reinhardtii PAM gene encodes an active peptide-amidating enzyme (CrPAM) that shares key structural and functional features with the mammalian enzyme, indicating that components of the peptide biosynthetic pathway predate multicellularity. In addition to its secretory pathway localization, CrPAM localizes to cilia and tightly associates with the axonemal superstructure, revealing a new axonemal enzyme activity. This localization pattern is conserved in mammals, with PAM present in both motile and immotile sensory cilia. The conserved ciliary localization of PAM adds to the known signaling capabilities of the eukaryotic cilium and provides a potential mechanistic link between peptidergic signaling and endocrine abnormalities commonly observed in ciliopathies.