Maxence V. Nachury

Cilia-derived extracellular vesicles (EVs) mediate cell-cell communication and remove signaling molecules from cilia. The Hedgehog receptor Smoothened (SMO) is selected for packaging into ciliary EVs by ubiquitin chains, but how cargo selection is coupled to EV budding remains unknown. In this work, working in mouse kidney cells, we found that the ubiquitin reader endosomal sorting complex required for transport–0 (ESCRT-0) orchestrates both cargo selection and EV biogenesis. Unlike the conventional ESCRT pathway, SMO secretion did not require the ESCRT-III membrane scission machinery. Instead, ESCRT-0 condensed at the ciliary tip, where it clustered ubiquitinated SMO to drive EV biogenesis. Disrupting ESCRT-0 condensation blocked SMO secretion, and combining forced recruitment of ESCRT-0 with optogenetic clustering of SMO was sufficient to reconstitute EV budding. Thus, ESCRT-0 condensation and cargo clustering constitute a minimal module for ESCRT-III–independent reverse-topology budding.

Signaling receptors can exit cilia either by retrieval back into the cell or by secretion in extracellular vesicles (EVs), a process known as ectocytosis. The mechanisms that govern ectocytosis remain poorly understood. Here, we leverage quantitative proteomic profiling to identify the Hedgehog signaling receptor Smoothened (SMO) as a major cargo of cilia-derived EVs. Surprisingly, ligands that promote ciliary accumulation of SMO strongly suppressed its packaging into EVs, indicating that ectocytosis selectively packages specific conformational states of SMO. We further find that SMO packaged into EVs is extensively modified with K63-linked ubiquitin chains. Preventing SMO ubiquitination or selectively removing K63-linked ubiquitin chains from SMO markedly reduced its secretion into EVs and caused SMO accumulation within cilia. Together, these results identify K63-linked ubiquitin chains as a sorting signal for ciliary ectocytosis and indicate that ubiquitin-dependent packaging of inactive SMO contributes to the dynamic redistribution of SMO during Hedgehog signaling.

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed notable tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Unexpectedly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity.

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed striking tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Surprisingly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity.

The BBSome mediates the retrieval of ubiquitinated membrane proteins from cilia, but its physiological cargoes in photoreceptors remain largely unidentified. Here, we find that K63-linked ubiquitin (UbK63) chains accumulate in the outer segment (OS, equivalent of cilia) of Bbs4 ^−/− photoreceptors from the onset of OS formation. Through quantitative profiling of the UbK63-associated OS proteome, we identify the transmembrane fragment of interphotoreceptor matrix proteoglycan 2 (IMPG2 ^m ) as a principal cargo of the BBSome. In Bbs4 ^−/− mice, ubiquitinated IMPG2 ^m aberrantly accumulates in OSs, and disruption of IMPG2 ^m ubiquitination impairs its retrieval and clearance. Because full-length IMPG2 traffics to the OS to deliver its extracellular domain to the matrix, our data support a model in which IMPG2 ^m undergoes constitutive cycling between the inner and outer segments. These findings redefine the BBSome’s role in photoreceptors from quality control to constitutive membrane protein turnover.

Structural homeostasis and proper distributions of signaling molecules in cilia require a constant flow of cargoes carried by intraflagellar transport (IFT) trains in both anterograde and retrograde directions within the thin, long ciliary shafts. In the motile cilium framework, the nine microtubule doublets of the same length serve as the transportation rails, and a preferential association to the two subtubules of the microtubule doublets prevents collisions among the IFT trains that move in opposite directions. However, this mechanism is incompatible with the primary cilia structure, where most of the nine microtubule doublets terminate in the ciliary shafts—only several of them reach the ciliary tip and only in a singlet form. Here, we demonstrate that anterograde and retrograde trains in primary cilia interact with both subtubules of the microtubule doublets without apparent preference. They can switch microtubules, and they may simultaneously interact with multiple microtubules to facilitate their movement. This architecture makes the collisions inevitable, and live-cell recordings reveal that anterograde and retrograde trains tend to pause when they come into direct contact. We also find that the velocity of the train’s movement often changes after the pause. Thus, the motion behaviors of IFT trains in primary cilia are distinctive from those of motile cilia, and our data offer an essential foundation for understanding proper signaling molecule distributions in primary cilia.

The G protein-coupled receptor (GPCR) melanocortin receptor 4 (MC4R) is an essential regulator of body weight homeostasis. MC4R is unusual among GPCRs in that its activity is regulated by 2 opposing physiological ligands, the agonist ⍺-MSH and the antagonist/inverse agonist AgRP. Paradoxically, while MC4R localizes and functions at the cilium of hypothalamic neurons, the ciliary levels of MC4R are very low under unrestricted feeding conditions. Here, we find that the constitutive activity of MC4R is responsible for the continuous depletion of MC4R from cilia and that inhibition of MC4R’s activity via AgRP leads to a robust accumulation of MC4R in cilia. Ciliary targeting of MC4R is mediated by its partner MRAP2 and the constitutive exit of MC4R from cilia relies on the sensor of activation β-arrestin, on ubiquitination, and on the BBSome ciliary trafficking complex. Thus, while MC4R exits cilia via conventional mechanisms, it only accumulates in cilia when its activity is suppressed by AgRP.

The G protein–coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor–associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.