Cilia Provide a Platform for the Generation, Regulated Secretion, and Reception of Peptidergic Signals.

Cilia are microtubule-based cellular projections that act as motile, sensory, and secretory organelles. These structures receive information from the environment and transmit downstream signals to the cell body. Cilia also release vesicular ectosomes that bud from the ciliary membrane and carry an array of bioactive enzymes and peptide products. Peptidergic signals represent an ancient mode of intercellular communication, and in metazoans are involved in the maintenance of cellular homeostasis and various other physiological processes and responses. Numerous peptide receptors, subtilisin-like proteases, the peptide-amidating enzyme, and bioactive amidated peptide products have been localized to these organelles. In this review, we detail how cilia serve as specialized signaling organelles and act as a platform for the regulated processing and secretion of peptidergic signals. We especially focus on the processing and trafficking pathways by which a peptide precursor from the green alga Chlamydomonas reinhardtii is converted into an amidated bioactive product-a chemotactic modulator-and released from cilia in ectosomes. Biochemical dissection of this complex ciliary secretory pathway provides a paradigm for understanding cilia-based peptidergic signaling in mammals and other eukaryotes.

Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.

Ciliary ectosomes are vesicles that bud from the ciliary membrane. Isolation and analysis of these structures can shed light on their bioactive cargoes and identify proteins and biomolecules involved in intercellular communication and various physiological processes. Most published methods to isolate ciliary ectosomes are based on their size (100nm to 1µm) to separate cilia-derived vesicles from isolated cilia and/or intact cells. However, it is often difficult to determine the origin of extracellular vesicles and to distinguish ciliary ectosomes from ectosomes budded from the plasma membrane or from exosomes that derive from multivesicular bodies. Here, we describe procedures to isolate and purify ciliary ectosomes from the unicellular green alga, Chlamydomonas reinhardtii, through differential and iodixanol density gradient ultracentrifugation; in this organism, the ciliary membrane is the only membrane directly exposed to the environment and thus ectosomes are of known origin. Ciliary ectosomes contain enzymes and α-amidated peptide products required to mediate peptidergic-signaling cascades; one identified amidated peptide acts as a chemotactic modulator for C. reinhardtii gametes. Classical methods used to assess chemotaxis do not provide quantitative measurements of the chemotactic gradient or the real-time effects on the migration of fast moving cells. Consequently, we developed a chemotaxis assay protocol using microfluidic channel slides that provides quantitative and qualitative measurements of the chemotactic gradient and cell migration. Here, we describe how to establish a stable gradient of a bioactive substance in microfluidic channel slides and perform quantitative assays to assess chemotaxis of both individual cells and populations of C. reinhardtii.

Regulated processing and secretion of a peptide precursor in cilia.

Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia-derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Peptidergic intercellular communication controls a wide range of physiological and behavioral responses and occurs throughout eukaryotes. The Chlamydomonas reinhardtii genome encodes what appear to be numerous prepropeptides and enzymes homologous to those used to convert metazoan prepropeptides into bioactive peptide products. Since C. reinhardtii, a green alga, lack the dense core vesicles in which metazoan peptides are processed and stored, we explored the hypothesis that propeptide processing and secretion occur through the regulated release of ciliary ectosomes. A synthetic peptide (GATI-amide) that could be generated from a 91-kDa peptide precursor (proGATI) serves as a chemotactic modulator, attracting minus gametes while repelling plus gametes. Here we dissect the processing pathway that leads to formation of an amidated peptidergic sexual signal specifically on the ciliary ectosomes of plus gametes. Unlike metazoan propeptides, modeling studies identified stable domains in proGATI. Mass spectrometric analysis of a potential prohormone convertase and the amidated proGATI-derived products found in cilia and mating ectosomes link endoproteolytic cleavage to ectosome entry. Extensive posttranslational modification of proGATI confers stability to its amidated product. Analysis of this pathway affords insight into the evolution of peptidergic signaling; this will facilitate studies of the secretory functions of metazoan cilia.

Cilia-derived vesicles: An ancient route for intercellular communication.

Extracellular vesicles (EVs) provide a mechanism for intercellular communication that transports complex signals in membrane delimited structures between cells, tissues and organisms. Cells secrete EVs of various subtypes defined by the pathway leading to release and by the pathological condition of the cell. Cilia are evolutionarily conserved organelles that can act as sensory structures surveilling the extracellular environment. Here we discuss the secretory functions of cilia and their biological implications. Studies in multiple species - from the nematode Caenorhabditis elegans and the chlorophyte alga Chlamydomonas reinhardtii to mammals - have revealed that cilia shed bioactive EVs (ciliary EVs or ectosomes) by outward budding of the ciliary membrane. The content of ciliary EVs is distinct from that of other vesicles released by cells. Peptides regulate numerous aspects of metazoan physiology and development through evolutionarily conserved mechanisms. Intriguingly, cilia-derived vesicles have recently been found to mediate peptidergic signaling. C. reinhardtii releases the peptide α-amidating enzyme (PAM), bioactive amidated products and components of the peptidergic signaling machinery in ciliary EVs in a developmentally regulated manner. Considering the origin of cilia in early eukaryotes, it is likely that release of peptidergic signals in ciliary EVs represents an alternative and ancient mode of regulated secretion that cells can utilize in the absence of dedicated secretory granules.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Cilia-based peptidergic signaling.

Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology.

Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia.

Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (AnkKI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in AnkKO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

Craniometaphyseal Dysplasia Mutations in ANKH Negatively Affect Human Induced Pluripotent Stem Cell Differentiation into Osteoclasts.

We identified osteoclast defects in craniometaphyseal dysplasia (CMD) using an easy-to-use protocol for differentiating osteoclasts from human induced pluripotent stem cells (hiPSCs). CMD is a rare genetic bone disorder, characterized by life-long progressive thickening of craniofacial bones and abnormal shape of long bones. hiPSCs from CMD patients with an in-frame deletion of Phe377 or Ser375 in ANKH are more refractory to in vitro osteoclast differentiation than control hiPSCs. To exclude differentiation effects due to genetic variability, we generated isogenic hiPSCs, which have identical genetic background except for the ANKH mutation. Isogenic hiPSCs with ANKH mutations formed fewer osteoclasts, resorbed less bone, expressed lower levels of osteoclast marker genes, and showed decreased protein levels of ANKH and vacuolar proton pump v-ATP6v0d2. This proof-of-concept study demonstrates that efficient and reproducible differentiation of isogenic hiPSCs into osteoclasts is possible and a promising tool for investigating mechanisms of CMD or other osteoclast-related disorders.

Expression of the SIN3 homologue from banana, MaSIN3, suppresses ABA responses globally during plant growth in Arabidopsis.

The SIN3 family of co-repressors is a family of highly conserved eukaryotic repressor proteins that regulates diverse functions in yeasts and animals but remains largely uncharacterized functionally even in plants like Arabidopsis. The sole SIN3 homologue in banana, MaSIN3, was identified as a 1408 amino acids, nuclear localized protein conserved to other SIN3s in the PAH, HID and HCR domains. Interestingly, MaSIN3 over-expression in Arabidopsis mimics a state of reduced ABA responses throughout plant development affecting growth processes such as germination, root growth, stomatal closure and water loss, flowering and senescence. The reduction in ABA responses is not due to reduced ABA levels but due to suppression of expression of several transcription factors mediating ABA responses. Transcript levels of negative regulators of germination (ABI3, ABI5, PIL5, RGL2 and RGL3) are reduced post-imbibition while those responsible for GA biosynthesis are up-regulated in transgenic MaSIN3 over-expressers. ABA-associated transcription factors are also down-regulated in response to ABA treatment. The HDAC inhibitors, SAHA and sodium butyrate, in combination with ABA differentially suppress germination in control and transgenic lines suggesting the recruitment by MaSIN3 of HDACs involved in suppression of ABA responses in different processes. The studies provide an insight into the ability of MaSIN3 to specifically affect a subset of developmental processes governed largely by ABA.

Synthesis and antimicrobial evaluation of ester-linked 1,4-disubstituted 1,2,3-triazoles with a furyl/thienyl moiety.

Twenty ester-linked 1,4-disubstituted 1,2,3-triazoles having a furyl/thienyl moiety have been synthesized from heteroaryl prop-2-yn-1-yl carboxylate and aromatic azides via a Cu(I) catalyzed 1,3-dipolar cycloaddition. All the synthesized compounds were characterized by FTIR, [Formula: see text]H NMR, [Formula: see text]C NMR spectroscopy and HRMS. Synthesized triazoles were tested in vitro for antimicrobial evaluation against Gram-negative bacteria-Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae; Gram-positive bacteria-Staphylococcus aureus and two fungal strains-Candida albicans and Aspergillus niger, reflecting moderate to good activity. The structure of compound 6f was also confirmed by X-ray crystallography (CCDC 1469326).