Primary cilium

The primary cilium is a sensory antenna-like organelle that extends as a solitary unit from the surface of nearly all vertebrate cell types. The presence of single non-motile cilia in vertebrate cells was first reported in the end of the 19th century by K.W. Zimmermann, who also suggested it to have a sensory role . The term “primary cilium” was later introduced by S. Sorokin. At the base of primary cilia is the basal body, which is formed from one of the centrioles of the centrosome after docking to the plasma membrane. Examples of proteins localizing to the primary cilium or the basal body can be seen in Figure 1.

In the subcellular resource, 653 genes (3% of all human protein-coding genes) have been shown to encode proteins that localize to primary cilia or basal bodies (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of genes encoding proteins in these compartments shows an enrichment of genes associated with biological processes related to assembly and organization of primary cilia, cell signaling and cell motility. Approximately 99% (n=648) of the proteins that localize to primary cilia or basal bodies can also be detected in additional cellular compartments, with the most common additional localizations being microtubules and centrosomes.


CFAP300 - hTERT-RPE1 (serum starved)

CKAP2 - ASC52telo

SMAD7 - RPTEC/TERT1

Figure 1. Examples of proteins localized to the cilium or basal body. CFAP300 is a cilium- and flagellum-specific protein that plays a role in axonemal structure organization and localizes specifically to the primary cilium. CKAP2 is a cytoskeleton-associated protein localizing to some cilia and engaging in cell-cycle regulation. SMAD7 is known to localize to the basal body and has been proposed to limit excessive GPCR signaling through controlling GPCR transport into and out of the cilium.

0%10%20%30%40%50%60%70%80%90%100%% genesNot mappedSecretedOther organellesPrimary cilium

  • 3% (653 proteins) of all human proteins have been experimentally detected in the primary cilium by the Human Protein Atlas.
  • 131 proteins in the primary cilium are supported by experimental evidence and out of these 3 proteins are enhanced by the Human Protein Atlas.
  • 648 proteins in the primary cilium have multiple locations.
  • 368 proteins in the primary cilium show single cell variation.

  • Proteins localizing to primary cilia are mainly involved in ciliognesis, cell signalling and cell motility.

Figure 2. 3% of all human protein-coding genes encode proteins that have been shown to localize to the nucleoplasm. Each bar is clickable and gives a search result of proteins that belong to the selected category.

The structure of the primary cilium

Substructures

  • Primary cilium: 384
  • Primary cilium tip: 55
  • Primary cilium transition zone: 88
  • Basal body: 401

Primary cilia are small antenna-like organelles that protrude from the surface of the majority of human cell types. In contrast to motile cilia, there is only one primary cilium in each cell. The primary cilium consist of a microtubule-based axoneme that extends the basal body, which is modified from the mother centriole of the centrosome, and is enclosed by a lipid bilayer. The microtubules of the axoneme forms a ring of nine doublets pairs, without any central pair as usually seen in motile cilia (9+0). However, recent studies indicate that this organisation is disrupted towards the tip of the cilium. The basal body is formed from the mother centriole and several additional protein components. It anchors the primary cilium to the cell body though pinwheel-shaped transition fibers (distal appendages), anchored to the membrane, and basal feet (subdistal appendages), anchchored to cytoplasmic microtubules. These are followed by a region referred to as the transition zone, in which the axonemal microtubules are connected to the ciliary membrane through multiple Y-links. The transition zone together with the transition fibers give rise to a selective barrier for diffusson of both soluble and membrane-bound proteins between the cell body and the primary cilium. Similarly, while the ciliary membrane is continuous with the plasma membrane, it has a distinct composition of lipids as well as proteins. The border between the plasma membrane and the primary cilium membrane is referred to as the periciliary membrane, and in some cell types it is invaginated to form a ciliary pocket (Mill P et al. (2023)).

The length of primary cilia varies extensively across the cell cycle, as well as between different cell types, ranging from few (e.g. chondrocytes) up to several tens of micrometres (e.g. kidney epithelial cells and neurons). In addition, there is evidence that the length of the primary cilia can be modified in response to specific environmental cues (Macarelli V et al. (2023)).

Primary cilia appear on non-dividing differentiated cells but also on stem and progenitor cells that are in the G1/G0 phases of the cell cycle (Satir P et al. (2010)). Assembly, elongation and disassembly is highy coordinated with the cell cycle. Before cell division, primary cilia are dismantled and the basal body is released to assume its role as a centriole during mitotic spindle assembly. New primary cilia are built again on each daughter cell once the cells are in the G1 or G0 phase. At the beginning of ciliogenesis the mother centriole migrates to the cell surface, docks onto the plasma membrane and transitions to a basal body. Then microtubules nucleate at the basal body to initiate the formation of the axoneme. For mesenchymal cells, formation of the ciliary membrane starts with fusion Golgi-derived vesicles to the mother centriole already before docking to the plasma membrane. For kidney epithelial cells, assembly of the ciliary membrane is initiated after docking at the plasma membrane. Proteins and lipids needed for furter assembly and elongation of the primary cilia membrane are in both cases transported in vesicles from the ER an Golgi compartments, and incorporated at the ciliary base by exocytosis.

Recent studies have shown that primary cilia can be highly enriched in specific signaling receptors, ion channels, and downstream effectors, for a growing number of pathways, including for example Hedgehog and WNT signalling. The protein and lipid composition of primary cilia varies across different cell types, tissues and can change in response to external developmental and homeostatic stimuli. This structural and compositional diversity requires tight control of the transport of both lipids and proteins into and out of the primary cilium, which is mediated by vesicular transport pathways as well as the intraflagellar transport (IFT) system (Taschner M et al. (2016)). The latter involves IFT-protein complexes, various adaptors, as well as microtubule-based motor proteins. In addition, lipid composition is regulated by cilia-residing enzymes.

Table 1. Selection of proteins suitable as markers for the primary cilium, primary cilium transition zone, or basal body.

Gene
Description
Substructure
ARL13B ADP ribosylation factor like GTPase 13B Acrosome
Basal body
Cytokinetic bridge
End piece
Equatorial segment
Microtubules
Mid piece
Primary cilium
Primary cilium transition zone
Principal piece
CFAP300 Cilia and flagella associated protein 300 Centrosome
Primary cilium
CEP350 Centrosomal protein 350 Basal body
Centrosome
End piece
Nucleoplasm
CEP164 Centrosomal protein 164 Centrosome
Equatorial segment
Nucleoplasm
Primary cilium transition zone
Principal piece
Vesicles


CFAP300 - hTERT-RPE1 (serum starved)

CFAP300 - RPTEC/TERT1

ARL13B - ASC52telo

CEP350

CEP164 - hTERT-RPE1 (serum starved)

KIF7 - ASC52telo

Figure 3. Examples showing the different staining patterns of primary cilia and basal bodies. CFAP300 is a cilium and flagellum specific protein (detected in hTERT-RPE1). ARL13B is involved in regulating the transport of certain proteins to cilia, it is present in the cytoplasm and cilium (detected in ASC52telo). CEP350 is a centrosome associated protein and since the centrosome is the core structure of the basal body, basal bodies are positive for CEP350. CEP164 is known as a part of the distal appendages of the centriole localizing in the region of the ciliary transition zone. KIF7 is in many cilia enriched at the tip of primary cilia.

The function of the primary cilium

In recent years, it has become more and more evident that primary cilia have an important role in detecting, regulating and transducing various types of information from the extracellular environment. Primary cilia are highly enriched in receptors, downstream effectors of various signaling pathways and ion channels. Depending on the spatiotemporal composition and localization of these components, they can detect and transduce a variety of signals, acting either as chemo-, mechano-, osmo- or photosensors (Nachury MV et al. (2019); Satir P et al. (2010)). The dynamic nature of the ciliary proteome may thus be important for cellular adaptation to specific developmental/homeostatic cues, thus ensuring efficient signal transduction in diverse developmental and homeostatic settings.

Primary cilia orchestrate a variety of signaling pathways (e.g. GPCR and WNT pathways) in order to regulate key developmental processes, as well as tissue plasticity and organ function in adulthood. Mutations that lead to impairment in the structure and/or function of primary cilia, or motile cilia, are associated with a group of more than 30 diseases and syndromes collectively known as ciliopathies. Ciliopathies span an overlapping and highly diverse spectrum of clinical symptoms, affecting a diverse set of organs, and are highly variable in terms of severity (Reiter JF et al. (2017); Anvarian Z et al. (2019)). Some well-studied examples of ciliopathies are polycystic kidney disease and the Bardet-Biedl syndrome, of which the latter is higly pleiotropic and shows variable expressivity even between individual patients. There are even examples in which different mutations of the same gene give rise to distinct ciliopathies demonstrating the complexity of these diseases.

Gene Ontology (GO) analysis of genes encoding proteins mainly localized to primary clia and basal bodies are well in agreement with a role for primary cilia in cell signalling pathways. The enriched terms for the GO domain Biological Process are mainly related to the assembly and organization of primary cilia, but also to cell signalling and cell motility (Figure 5a). Enrichment analysis of the GO domain Molecular Function, gives enrichment of terms related to different categories of protein binding activity and motor protein activity (Figure 5b).

051015202530354045Fold EnrichmentMicrotubule anchoring at cen...Protein localization to ciliumNon-motile cilium assemblyCilium organizationCiliary basal body-plasma me...Convergent extensionResponse to laminar fluid sh...Protein localization to cent...Protein localization to micr...Photoreceptor cell maintenanceSmoothened signaling pathwayCilium or flagellum-dependen...Cilium-dependent cell motilityMicrotubule-based transportOrganelle localization by me...Membrane dockingPhotoreceptor cell developmentFlagellated sperm motilitySperm motilityRegulation of G2/M transitio...Regulation of plasma membran...Establishment of cell polarityRegulation of G protein-coup...Visual perceptionUrogenital system developmentVisual system developmentShow full plot

Figure 5a Gene Ontology-based enrichment analysis for the primany cilium and basal body proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.

024681012141618202224Fold EnrichmentDynein heavy chain bindingProtein kinase A catalytic s...3',5'-cyclic-GMP phosphodies...Dynein light intermediate ch...ATP-dependent microtubule mo...Dynein light chain bindingDynein intermediate chain bi...Microtubule motor activityTubulin bindingGuanyl nucleotide bindingGuanyl ribonucleotide binding

Figure 5b Gene Ontology-based enrichment analysis for the primany cilium and basal body proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.

Primary cilium proteins with multiple locations

In the subcellular resource, approximately 99% (n=648) of the proteins that localize to primary cilia or basal bodies also localize to other cell compartments (Figure 6). The network plot shows that the most common locations shared with primary cilia are the centrosome, cytosol and microtubules. Dual localizations of primary cilium proteins to the centrosome or microtubules are overrepresented, which is expected based on the structure of primary cilia and basal bodies. Examples of multilocalizing proteins within the nucleoplasmic proteome can be seen in Figure 7.

Figure 6. Interactive network plot of primary cilia and basal body proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to primary cilia or basal bodies, and to one or more additional locations. Only connecting nodes containing at least 1% of the proteins in the primary cilia- and basal body proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.


AKT1 - ASC52telo

CKAP2 - ASC52telo

CEP170 - ASC52telo

Figure 7. Examples of multilocalizing proteins in primary cilia and basal bodies. The examples show common or overrepresented combinations for multilocalizing proteins in the primary cilia and basal body proteome. AKT1 is found both at primary cilia and microtubules.CKAP2 localizes to primary cilia and the mitotic spindle. CEP170 is seen both at basal bodies and centrosomes.

Expression levels of primary cilium proteins in tissue

Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that genes encoding proteins localizing to primary cilia and basal bodies have a similar distrubution across these categories as all genes with data in the subcellular resource, except that significantly fewer are undetected.

*Detected in singleDetected in someDetected in manyDetected in allNot detected0.0102030405060708090100%Primary ciliumAll localized genes

Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for nuclear protein-coding genes compared to all genes in the subcellular sesource. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.

Relevant links and publications

Mill P et al., Primary cilia as dynamic and diverse signalling hubs in development and disease. Nat Rev Genet. (2023)
PubMed: 37072495 DOI: 10.1038/s41576-023-00587-9

Macarelli V et al., Regulation of the length of neuronal primary cilia and its potential effects on signalling. Trends Cell Biol. (2023)
PubMed: 37302961 DOI: 10.1016/j.tcb.2023.05.005

Satir P et al., The primary cilium at a glance. J Cell Sci. (2010)
PubMed: 20144997 DOI: 10.1242/jcs.050377

Taschner M et al., The Intraflagellar Transport Machinery. Cold Spring Harb Perspect Biol. (2016)
PubMed: 27352625 DOI: 10.1101/cshperspect.a028092

Nachury MV et al., Establishing and regulating the composition of cilia for signal transduction. Nat Rev Mol Cell Biol. (2019)
PubMed: 30948801 DOI: 10.1038/s41580-019-0116-4

Reiter JF et al., Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. (2017)
PubMed: 28698599 DOI: 10.1038/nrm.2017.60

Anvarian Z et al., Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol. (2019)
PubMed: 30733609 DOI: 10.1038/s41581-019-0116-9