The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60/DYNC2I1 and the DYNC2H1–DYNC2LI1 dimer from dynein-2, and IFT54 and IFT57 from IFT-B contribute to the dynein-2–IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.

Cytoplasmic dynein-2 is a motor protein complex that drives the movement of cargoes along microtubules within cilia, facilitating the assembly of these organelles on the surface of nearly all mammalian cells. Dynein-2 is crucial for ciliary function, as evidenced by deleterious mutations in patients with skeletal abnormalities. Longstanding questions include how the dynein-2 complex is assembled, regulated, and switched between active and inactive states. A combination of model organisms, in vitro cell biology, live-cell imaging, structural biology and biochemistry has advanced our understanding of the dynein-2 motor. In this Cell Science at a Glance article and the accompanying poster, we discuss the current understanding of dynein-2 and its roles in ciliary assembly and function.

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway.

During cerebellum development, Sonic hedgehog (Shh)-induced proliferation of cerebellar granular neuronal precursors (CGNPs) is potently inhibited by bone morphogenetic proteins (BMPs). We have previously reported the upregulation of TIEG-1 and Mash1, two antimitotic factors that modulate MYCN transcription and N-Myc activity, in response to BMP2. To gain further insight into the BMP antimitotic mechanism, we used microRNA (miRNA) arrays to compare the miRNAs of CGNPs proliferating in response to Shh with those of CGNPs treated with Shh plus BMP2. The array analysis revealed that miRNA 11 (miR-22) levels significantly increased in cells treated with BMP2. Additionally, in P7 mouse cerebellum, miR-22 distribution mostly recapitulated the combination of BMP2 and BMP4 expression patterns. Accordingly, in CGNP cultures, miR-22 overexpression significantly reduced cell proliferation, whereas miR-22 suppression diminished BMP2 antiproliferative activity. In contrast to BMP2, miR-22 did not induce neural differentiation but instead significantly increased cell cycle length. Consistent with the central role played by N-myc on CGNP proliferation, Max was revealed as a direct target of miR-22, and miR-22 expression caused a significant reduction of Max protein levels and N-myc/Max-dependent promoter activity. Therefore, we conclude that, in addition to the previously described mechanisms, miR-22 plays a specific role on downstream BMPs through cerebellum growth.

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. Motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport respectively in the cilium. WDR34, a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome or asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34 knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity, and Hedgehog signalling were also affected.

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.