Comparative ultrastructure of the olfactory system in the East African root rat (Tachyoryctes splendens) and the naked mole rat (Heterocephalus glaber).

The ultrastructure of the olfactory system of most fossorial rodents remains largely unexplored. This study sought to investigate the functional structure of the olfactory mucosa and olfactory bulb of two species of fossorial rodents that have distinct behaviour and ecology, the East African root rat (RR) and the naked mole rat (NMR). Transmission electron microscopy and scanning electron microscopy were employed. The basic ultrastructural design of the olfactory system of the two species was largely comparable. In both species, the olfactory mucosa comprised an olfactory epithelium and an underlying lamina propria. The olfactory epithelium revealed olfactory knobs, cilia and microvilli apically and sustentancular cells, olfactory receptor neurons and basal cells in the upper, middle and basal zones, respectively. The lamina propria was constituted by Bowman's glands, olfactory nerve bundles and vasculature supported by loose connective tissue. Within the olfactory bulb, intracellular and extracellular structures including cell organelles, axons and dendrites were elucidated. Notable species differences were observed in the basal zone of the olfactory epithelium and on the luminal surface of the olfactory mucosa. The basal zone of the olfactory epithelium of the RR consisted of a single layer of flattened electron-dense horizontal basal cells while the NMR had juxtaposed electron-dense and electron-lucent heterogenous cells, an occurrence seen as being indicative of quiescent and highly proliferative states of the olfactory epithelia in the two species, respectively. The olfactory epithelial surface of the NMR comprised an elaborate cilia network that intertwined extensively forming loop-like structures whereas in the RR, the surface was rugged and consisted of finger-like processes and irregular masses. With gross and histological studies showing significant differences in the olfactory structures of the two species, these findings are a further manifestation that the olfactory system of the RR and the NMR have evolved differently to reflect their varied olfactory functional needs.

The MBO2/FAP58 heterodimer stabilizes assembly of inner arm dynein b and reveals axoneme asymmetries involved in ciliary waveform.

Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.

BACKGROUND: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway. RESULTS: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity. CONCLUSIONS: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

Bioinformatic validation and machine learning-based exploration of purine metabolism-related gene signatures in the context of immunotherapeutic strategies for nonspecific orbital inflammation.

Background: Nonspecific orbital inflammation (NSOI) represents a perplexing and persistent proliferative inflammatory disorder of idiopathic nature, characterized by a heterogeneous lymphoid infiltration within the orbital region. This condition, marked by the aberrant metabolic activities of its cellular constituents, starkly contrasts with the metabolic equilibrium found in healthy cells. Among the myriad pathways integral to cellular metabolism, purine metabolism emerges as a critical player, providing the building blocks for nucleic acid synthesis, such as DNA and RNA. Despite its significance, the contribution of Purine Metabolism Genes (PMGs) to the pathophysiological landscape of NSOI remains a mystery, highlighting a critical gap in our understanding of the disease's molecular underpinnings. Methods: To bridge this knowledge gap, our study embarked on an exploratory journey to identify and validate PMGs implicated in NSOI, employing a comprehensive bioinformatics strategy. By intersecting differential gene expression analyses with a curated list of 92 known PMGs, we aimed to pinpoint those with potential roles in NSOI. Advanced methodologies, including Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA), facilitated a deep dive into the biological functions and pathways associated with these PMGs. Further refinement through Lasso regression and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) enabled the identification of key hub genes and the evaluation of their diagnostic prowess for NSOI. Additionally, the relationship between these hub PMGs and relevant clinical parameters was thoroughly investigated. To corroborate our findings, we analyzed expression data from datasets GSE58331 and GSE105149, focusing on the seven PMGs identified as potentially crucial to NSOI pathology. Results: Our investigation unveiled seven PMGs (ENTPD1, POLR2K, NPR2, PDE6D, PDE6H, PDE4B, and ALLC) as intimately connected to NSOI. Functional analyses shed light on their involvement in processes such as peroxisome targeting sequence binding, seminiferous tubule development, and ciliary transition zone organization. Importantly, the diagnostic capabilities of these PMGs demonstrated promising efficacy in distinguishing NSOI from non-affected states. Conclusions: Through rigorous bioinformatics analyses, this study unveils seven PMGs as novel biomarker candidates for NSOI, elucidating their potential roles in the disease's pathogenesis. These discoveries not only enhance our understanding of NSOI at the molecular level but also pave the way for innovative approaches to monitor and study its progression, offering a beacon of hope for individuals afflicted by this enigmatic condition.

Advancing Primary Ciliary Dyskinesia Diagnosis through High-Speed Video Microscopy Analysis.

Primary ciliary dyskinesia (PCD) is an inherited disorder that impairs motile cilia, essential for respiratory health, with a reported prevalence of 1 in 16,309 within Hispanic populations. Despite 70% of Puerto Rican patients having the RSPH4A [c.921+3_921+6del (intronic)] founder mutation, the characterization of the ciliary dysfunction remains unidentified due to the unavailability of advanced diagnostic modalities like High-Speed Video Microscopy Analysis (HSVA). Our study implemented HSVA for the first time on the island as a tool to better diagnose and characterize the RSPH4A [c.921+3_921+6del (intronic)] founder mutation in Puerto Rican patients. By applying HSVA, we analyzed the ciliary beat frequency (CBF) and pattern (CBP) in native Puerto Rican patients with PCD. Our results showed decreased CBF and a rotational CBP linked to the RSPH4A founder mutation in Puerto Ricans, presenting a novel diagnostic marker that could be implemented as an axillary test into the PCD diagnosis algorithm in Puerto Rico. The integration of HSVA technology in Puerto Rico substantially enhances the PCD evaluation and diagnosis framework, facilitating prompt detection and early intervention for improved disease management. This initiative, demonstrating the potential of HSVA as an adjunctive test within the PCD diagnostic algorithm, could serve as a blueprint for analogous developments throughout Latin America.

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids.

Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns. Biliatresone, a plant toxin, causes BA-like syndrome in some animals, but its relevance in humans is unknown. To validate the hypothesis that biliatresone exposure is a plausible BA disease mechanism in humans, we treated normal human liver organoids with biliatresone and addressed its adverse effects on organoid development, functions and cellular organization. The control organoids (without biliatresone) were well expanded and much bigger than biliatresone-treated organoids. Expression of the cholangiocyte marker CK19 was reduced, while the hepatocyte marker HFN4A was significantly elevated in biliatresone-treated organoids. ZO-1 (a tight junction marker) immunoreactivity was localized at the apical intercellular junctions in control organoids, while it was markedly reduced in biliatresone-treated organoids. Cytoskeleton F-actin was localized at the apical surface of the control organoids, but it was ectopically expressed at the apical and basal sides in biliatresone-treated organoids. Cholangiocytes of control organoids possess primary cilia and elicit cilia mechanosensory function. The number of ciliated cholangiocytes was reduced, and cilia mechanosensory function was hampered in biliatresone-treated organoids. In conclusion, biliatresone induces morphological and developmental changes in human liver organoids resembling those of our previously reported BA organoids, suggesting that environmental toxins could contribute to BA pathogenesis.

Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function.

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.

Primary Cilium in Neural Crest Cells Crucial for Anterior Segment Development and Corneal Avascularity.

Purpose: Intraflagellar transport 46 (IFT46) is an integral subunit of the IFT-B complex, playing a key role in the assembly and maintenance of primary cilia responsible for transducing signaling pathways. Despite its predominant expression in the basal body of cilia, the precise role of Ift46 in ocular development remains undetermined. This study aimed to elucidate the impact of neural crest (NC)-specific deletion of Ift46 on ocular development. Methods: NC-specific conditional knockout mice for Ift46 (NC-Ift46F/F) were generated by crossing Ift46F mice with Wnt1-Cre2 mice, enabling the specific deletion of Ift46 in NC-derived cells (NCCs). Sonic Hedgehog (Shh) and Notch signaling activities in NC-Ift46F/F mice were evaluated using Gli1lacZ and CBF:H2B-Venus reporter mice, respectively. Cell fate mapping was conducted using ROSAmTmG reporter mice. Results: The deletion of Ift46 in NCCs resulted in a spectrum of ocular abnormalities, including thickened corneal stroma, hypoplasia of the anterior chamber, irregular iris morphology, and corneal neovascularization. Notably, this deletion led to reduced Shh signal activity in the periocular mesenchyme, sustained expression of key transcription factors Foxc1, Foxc2 and Pitx2, along with persistent cell proliferation. Additionally, it induced increased Notch signaling activity and the development of ectopic neovascularization within the corneal stroma. Conclusions: The absence of primary cilia due to Ift46 deficiency in NCCs is associated with anterior segment dysgenesis (ASD) and corneal neovascularization, suggesting a potential link to Axenfeld-Rieger syndrome, a disorder characterized by ASD. This underscores the pivotal role of primary cilia in ensuring proper anterior segment development and maintaining an avascular cornea.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

Comparing region of interest selection and whole-field analysis for measurement of ciliary beat frequency in high-speed video analysis.

BACKGROUND: Ciliary beat frequency (CBF) is crucial in mucociliary clearance. High-speed video analysis (HSVA) is commonly used to measure CBF but lacks standardization. We compared visual observation and computer-assisted calculation using fast Fourier transformation (FFT) in freshly collected bronchial ciliary epithelial cells and cultured cells. METHODS: Bronchial epithelial cells were obtained from 12 patients who required bronchoscopic examination. Eighty-five videos of ciliary movement of freshly collected and cultured cells were recorded and used to calculate CBF using manual observation, region of interest (ROI) selection, and whole-field analysis. RESULTS: CBF measured by the ROI selection method strongly correlated with that measured using manual observation, especially in freshly collected cells. However, 27.8% of the manual observation method values were doubled in the ROI selection method, probably because a round trip of cilia was calculated as two cycles and needed to be corrected to 1/2 value. Upon increasing the number of ROIs, the results of the ROI selection method came closer to that of WFA. CONCLUSIONS: Computer-assisted calculation using FFT can aid in measuring CBF; however, current methods require visual confirmation. Further automated evaluation techniques are needed to establish more standardized and generalized CBF measurement methods using HSVA.

Structural determination and modeling of ciliary microtubules.

The axoneme, a microtubule-based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo-EM and cryo-ET have allowed a molecular-level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo-EM and cryo-ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI-guided tools that are transforming structural biology.

Molecular basis promoting centriole triplet microtubule assembly.

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.

Positioning centrioles and centrosomes.

Centrosomes are the primary microtubule organizer in eukaryotic cells. In addition to shaping the intracellular microtubule network and the mitotic spindle, centrosomes are responsible for positioning cilia and flagella. To fulfill these diverse functions, centrosomes must be properly located within cells, which requires that they undergo intracellular transport. Importantly, centrosome mispositioning has been linked to ciliopathies, cancer, and infertility. The mechanisms by which centrosomes migrate are diverse and context dependent. In many cells, centrosomes move via indirect motor transport, whereby centrosomal microtubules engage anchored motor proteins that exert forces on those microtubules, resulting in centrosome movement. However, in some cases, centrosomes move via direct motor transport, whereby the centrosome or centriole functions as cargo that directly binds molecular motors which then walk on stationary microtubules. In this review, we summarize the mechanisms of centrosome motility and the consequences of centrosome mispositioning and identify key questions that remain to be addressed.

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite.

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

Biomechanics of transduction by mechanosensory cilia for prey detection in aquatic organisms.

Surface-feeding aquatic animals navigate towards the source of water disturbances and must differentiate prey from other environmental stimuli. Medicinal leeches locate prey, in part, using a distribution of mechanosensory hairs along their body that deflect under fluid flow. Leech's behavioral responses to surface wave temporal frequency are well documented. However, a surface wave's temporal frequency depends on many underlying environmental and fluid properties that vary substantially in natural habitats (e.g., water depth, temperature). The impact of these variables on neural response and behavior is unknown. Here, we developed a physics-based leech mechanosensor model to examine the impact of environmental and fluid properties on neural response. Our model used the physical properties of a leech cilium and was verified against existing behavioral and electrophysiological data. The model's peak response occurred with waves where the effects of gravity and surface tension were nearly equal (i.e., the phase velocity minimum). This suggests that preferred stimuli are related to the interaction between fundamental properties of the surrounding medium and the mechanical properties of the sensor. This interaction likely tunes the sensor to detect the nondispersive components of the signal, filtering out irrelevant ambient stimuli, and may be a general property of cilia across the animal kingdom.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.

BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function. METHODS: The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ. RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP. CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.

The CPLANE protein Fuzzy regulates ciliogenesis by suppressing actin polymerization at the base of the primary cilium via p190A RhoGAP.

The primary cilium decorates most eukaryotic cells and regulates tissue morphogenesis and maintenance. Structural or functional defects of primary cilium result in ciliopathies, congenital human disorders affecting multiple organs. Pathogenic variants in the ciliogenesis and planar cell polarity effectors (CPLANE) genes FUZZY, INTU and WDPCP disturb ciliogenesis, causing severe ciliopathies in humans and mice. Here, we show that the loss of Fuzzy in mice results in defects of primary cilia, accompanied by increased RhoA activity and excessive actin polymerization at the basal body. We discovered that, mechanistically, Fuzzy interacts with and recruits the negative actin regulator ARHGAP35 (also known as p190A RhoGAP) to the basal body. We identified genetic interactions between the two genes and found that a mutant ArhGAP35 allele increases the severity of phenotypic defects observed in Fuzzy-/- mice. Based on our findings, we propose that Fuzzy regulates ciliogenesis by recruiting ARHGAP35 to the basal body, where the latter likely restricts actin polymerization and modifies the actin network. Our study identifies a mechanism whereby CPLANE proteins control both actin polymerization and primary cilium formation.

Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.