In situ proximity ligation assay for analysing spatial interactions between ciliary proteins

Modeling Human Disease in Humans: The Ciliopathies

To understand cellular processes and events responsible for their perturbations, proteomic analyses are needed in bio-medical research and clinical diagnostics. Several techniques based on specifically binding reagents (antibodies) or recombinant proteins (GFP fusion protein, methods of fluorescence/ bio-luminescence resonance energy transfer) are generally used to study protein location and activity resulting from secondary modifications and interactions. The in situ proximity ligation assay represents a novel technique of in situ protein imaging using DNA as a reporter molecule and DNA amplification processes. This method enables direct visualization of single molecules, their levels, modifications and pattern of interactions in individual fixed cells and tissues. Proximity probes consist of specific antibody with attached oligonucleotides that are used as reporter molecules for identification of such events. Proximity probes guide the formation of a circular DNA strand when bound in close proximity. The DNA circle after that serves as a template for rolling circle amplification allowing the interaction to be visualized. Compared to available proteomic techniques benefiting from genetic engineering, in situ PLA enables study of endogenous proteins in their natural environment and thus can be used for clinical specimens. The areas of applicability where proximity ligation procedure can be used include any research field where protein interaction measurements are important, such as signal-ing pathway studies, monitoring of pharmacological treatment targets and oncological diagnostics.

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia

The Emerging Complexity of the Vertebrate Cilium: New Functional Roles for an Ancient Organelle

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Iron forms essential cofactors used by many nuclear enzymes involved in genome maintenance. However, unchaperoned nuclear iron may represent a threat to the surrounding genetic material as it promotes redox toxicity that may affect DNA integrity. Safely handling intracellular iron implies metal transfer and cofactor assembly processes based on protein-protein interactions. Identifying those interactions commonly occurs via high-throughput approaches using affinity purification or proximity labeling coupled with mass spectrometry analysis. However, these methods do not identify the subcellular location of the interactions. The one-on-one confirmation of proposed nuclear interactions is also challenging. Many approaches used to look at protein interactions are not tailored for looking at the nucleus because the methods used to solubilize nuclear content are harsh enough to disrupt those transient interactions. Here, we describe step-by-step the use of Proximity Ligation Assay (PLA) to analyze iron-mediated protein-protein interactions in the nucleus of cultured human cells. PLA allows the subcellular visualization of the interactions via the in situ detection of the two interacting proteins using fluorescence confocal microscopy. Briefly, cells are fixed, blocked, permeabilized, and incubated with primary antibodies directed to target proteins. Primary antibodies are recognized using PLA probes consisting of one PLUS and one MINUS oligonucleotide-labeled secondary antibody. If the two proteins are close enough (<40nm), the PLA probes are ligated and used as the template for rolling circle amplification (RCA) with fluorescently labeled oligonucleotides that yield a signal detectable using fluorescence confocal microscopy. A fluorescently labeled membrane-specific stain (WGA) and the DNA-specific probe DAPI are used to identify cellular and nuclear boundaries, respectively. Confocal images are then analyzed using the CellProfiler software to confirm the abundance and localization of the studied protein-protein interactions.

The transition zone (TZ) of the cilium/flagellum serves as a diffusion barrier that controls the entry/exit of ciliary proteins. Mutations of the TZ proteins disrupt barrier function and lead to multiple human diseases. However, the systematic regulation of ciliary composition and signaling-related processes by different TZ proteins is not completely understood. Here, we reveal that loss of TCTN1 in Chlamydomonas reinhardtii disrupts the assembly of wedge-shaped structures in the TZ. Proteomic analysis of cilia from WT and three TZ mutants, tctn1, cep290, and nphp4, shows a unique role of each TZ subunit in the regulation of ciliary composition, explaining the phenotypic diversity of different TZ mutants. Interestingly, we find that defects in the TZ impair the formation and biological activity of ciliary ectosomes. Collectively, our findings provide systematic insights into the regulation of ciliary composition by TZ proteins and reveal a link between the TZ and ciliary ectosomes.

BackgroundCilia are microtubule-based organelles present on the surface of many eukaryotic cell types critical for tissue homeostasis and proper organ development. Ciliary dysfunction underlies a growing list of human diseases and disorders collectively called ciliopathies such as Bardet-Biedl syndrome (BBS), Joubert syndrome, Meckel–Gruber syndrome and primary ciliary dyskinesia. Many ciliary proteins are associated with neuronal function consistent with neuronal developmental delays, cognitive, learning, and memory deficits observed in several ciliopathies, suggesting that ciliary dysfunction may contribute to pathogenesis of neuronal diseases and that an understanding of how ciliary proteins function together as a system would provide much needed mechanistic insights into their molecular etiologies.MethodsWe constructed protein-protein interaction (PPI) networks of genes associated with cilia and those associated with 7 neuropsychiatric diseases: schizophrenia, attention deficit hyperactivity disorder, major depressive disorder, bipolar disorder, autism spectrum disorder, Alzheimer’s disease and Parkinson’s disease. The interactome is constructed with experimentally determined PPIs from BioGRID and HPRD databases and novel PPIs predicted using our High-confidence PPI Prediction (HiPPIP) model. We previously presented Schizophrenia Interactome constructed using HiPPIP andalso showed that novel PPIs are highly accurate based on computational and experimental validations. We validated additional PPIs of cilia interactome here. We computed how closely connected cilia is to genes associated with neuropsychiatric diseases, through interactome and pathway analysis. Additionally, we analyzed drugs that proteins in the cilia interactome, and found that majority of these drugs are nervous system associated drugs.ResultsThe ciliary protein interactome consists of 165 ciliary proteins with 1,011 known PPIs and 765 novel PPIs. We found the overlap between cilia and neuropsychiatric interactomes to be statistically highly significant. For e.g., cilia interactome has an overlap of 125 genes with schizophrenia interactome of which 26 are novel interactors of cilia, and has significant overlap with pathways relevant to schizophrenia. About 184 genes in the cilia interactome are targeted by 548 FDA approved drugs, of which 103 are used to treat nervous system diseases.DiscussionCiliary genes like DRD1 and DRD2 are implicated in neurotransmission and associated with schizophrenia. DRD1 has 4 novel interactors and DRD2 has 12 novel interactors that may have significant role in the pathology of mental disorders. Neuronal pathways associated with cilia interactome with high statistical significance such as dopamine signaling, eNOS signaling, synaptic long-term potentiation pathways are known to be associated schizophrenia. Wnt signaling and PCP signaling are also known to be associated with cilia mediated neurodevelopmental signaling, defects in these pathways contributing to schizophrenia. Novel interactions for cilia proteins validated by experiments have functional significance in association with cilia and neuronal disorders. For e.g., IFT88, a cilia protein required for cilia assembly, is critical for SHH signaling, cell cycle regulation and cerebellar development and is also associated with schizophrenia and bipolar disorder. CACNA1I is predicted to interact with DNAL4 and MKS1, both involved in transport of proteins required for ciliogenesis. GWAS studies show that CACNA1I is associated with schizophrenia. Taken together, the cilia interactome presented here provides novel insights into the relationship between ciliary protein function and neuropsychiatric diseases.

Cilia are dynamic microtubule-based organelles present on the surface of many eukaryotic cell types and can be motile or non-motile primary cilia. Cilia defects underlie a growing list of human disorders, collectively called ciliopathies, with overlapping phenotypes such as developmental delays and cognitive and memory deficits. Consistent with this, cilia play an important role in brain development, particularly in neurogenesis and neuronal migration. These findings suggest that a deeper systems-level understanding of how ciliary proteins function together may provide new mechanistic insights into the molecular etiologies of nervous system defects. Towards this end, we performed a protein–protein interaction (PPI) network analysis of known intraflagellar transport, BBSome, transition zone, ciliary membrane and motile cilia proteins. Known PPIs of ciliary proteins were assembled from online databases. Novel PPIs were predicted for each ciliary protein using a computational method we developed, called High-precision PPI Prediction (HiPPIP) model. The resulting cilia “interactome” consists of 165 ciliary proteins, 1,011 known PPIs, and 765 novel PPIs. The cilia interactome revealed interconnections between ciliary proteins, and their relation to several pathways related to neuropsychiatric processes, and to drug targets. Approximately 184 genes in the cilia interactome are targeted by 548 currently approved drugs, of which 103 are used to treat various diseases of nervous system origin. Taken together, the cilia interactome presented here provides novel insights into the relationship between ciliary protein dysfunction and neuropsychiatric disorders, for e.g. interconnections of Alzheimer’s disease, aging and cilia genes. These results provide the framework for the rational design of new therapeutic agents for treatment of ciliopathies and neuropsychiatric disorders.

Paramecium is a free-living unicellular organism, easy to cultivate, featuring ca. 4000 motile cilia emanating from longitudinal rows of basal bodies anchored in the plasma membrane. The basal body circumferential polarity is marked by the asymmetrical organization of its associated appendages. The complex basal body plus its associated rootlets forms the kinetid. Kinetids are precisely oriented within a row in correlation with the cell polarity. Basal bodies also display a proximo-distal polarity with microtubule triplets at their proximal ends, surrounding a permanent cartwheel, and microtubule doublets at the transition zone located between the basal body and the cilium. Basal bodies remain anchored at the cell surface during the whole cell cycle. On the opposite to metazoan, there is no centriolar stage and new basal bodies develop anteriorly and at right angle from the base of the docked ones. Ciliogenesis follows a specific temporal pattern during the cell cycle and both unciliated and ciliated docked basal bodies can be observed in the same cell. The transition zone is particularly well organized with three distinct plates and a maturation of its structure is observed during the growth of the cilium. Transcriptomic and proteomic analyses have been performed in different organisms including Paramecium to understand the ciliogenesis process. The data have incremented a multi-organism database, dedicated to proteins involved in the biogenesis, composition and function of centrosomes, basal bodies or cilia. Thanks to its thousands of basal bodies and the well-known choreography of their duplication during the cell cycle, Paramecium has allowed pioneer studies focusing on the structural and functional processes underlying basal body duplication. Proteins involved in basal body anchoring are sequentially recruited to assemble the transition zone thus indicating that the anchoring process parallels the structural differentiation of the transition zone. This feature offers an opportunity to dissect spatio-temporally the mechanisms involved in the basal body anchoring process and transition zone formation.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Ligation-mediated circuit-driven cascade amplification with high utilization rate of template for lncRNA MALAT1 detection in cancer mice tissues.

The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.

Introduction: Primary cilia are microtubule-based extensions of the plasma membrane in nearly all cell types. In vertebrate photoreceptors, the sensory cilium develops as outer segment (OS) that contains the photopigment rhodopsin and other proteins necessary for phototransduction. The distinct composition of proteins and lipids in the OS membrane is maintained by the selective barrier located at the border between the basal body and the ciliary compartment, called the transition zone (TZ).Areas covered: In this review, we will discuss the identification and function of two ciliary TZ proteins, RPGR (retinitis pigmentosa GTPase regulator) and CEP290. Mutations in these proteins account for a majority of retinopathies due to ciliary dysfunction. We will also discuss the potential of such information in designing therapeutic approaches to treat cilia-dependent photoreceptor degenerative diseases.Expert opinion: RPGR and CEP290 perform overlapping yet distinct functions in regulating trafficking of cargo via the TZ of photoreceptors. While RPGR modulates the trafficking by acting as a GEF for the small GTPase RAB8A, CEP290 may be involved in maintaining the polarized distribution of proteins in the OS by modulating intracellular levels of selected proteins involved in inhibiting OS formation.