Biology subjects

Mick, D. U.

Publications and source records attributed to Mick, D. U..

6 recordsLinked to original sources

bioRxiv2026

K63-linked ubiquitin chains mark inactive Smoothened for packaging into ciliary extracellular vesicles

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.

cell biology↗

bioRxiv2025

Proximity proteomics of primary cilia in human hypothalamic neurons

Primary cilia are hair-like sensory organelles that project from the cell bodies of most cell types, including appetite-regulatory hypothalamic neurons where they likely help sense metabolic factors to regulate food intake. We hypothesized that characterising the proteins present in the primary cilia of hypothalamic neurons would shed mechanistic insights into their sensory role and identify new therapeutic targets for obesity. We therefore targeted the ascorbate peroxidase APEX2 to primary cilia in human induced pluripotent stem cells (hiPSC)-derived hypothalamic neurons to biotinylate and identify ciliary proteins. Among the cilia-enriched proteins, we identified synaptic proteins, neurotransmitter receptors, and cell-cell adhesion and axon guidance proteins, extending recent findings that primary cilia interact with neuronal synapses. We also found genes associated with increased body weight and metabolic phenotypes that could represent new therapeutic targets including the lysophosphatidic receptor 1 (LPAR1), which we validated is cilia-localized and we confirmed that its ligand (LPA) mediates ciliary shortening. These findings provide insights into the molecular mechanisms by which primary cilia functionally impact appetite-regulatory neurons.

cell biology↗

bioRxiv2025

iAPEX: Improved APEX-based proximity labeling for subcellular proteomics using an enzymatic reaction cascade

Ascorbate peroxidase (APEX) is a versatile labeling enzyme used for live-cell proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. By combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide, we establish a more versatile, improved APEX (iAPEX) workflow that minimizes hydrogen peroxide toxicity and reduces non-specific background labeling. We employ iAPEX to perform live-cell proteomics of a cellular microdomain, the primary cilium, in previously inaccessible cell lines, leading to the identification of new ciliary proteins. Our study robustly validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. iAPEX proximity labeling in Xenopus laevis provides a proof-of-concept for future in vivo applications.

biochemistry↗

bioRxiv2022

The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia

Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as Hedgehog morphogens. Lysine 63-linked ubiquitin (K63Ub) chains mark GPCRs for regulated removal from cilia, but the molecular basis of K63Ub recognition inside cilia remains elusive. Here we show that the BBSome -the trafficking complex in charge of retrieving GPCRs from cilia- engages the ancestral endosomal sorting factor TOM1L2 (Target of Myb1-Like 2) to recognize UbK63 chains within cilia. TOM1L2 directly binds to UbK63 chains and to the BBSome and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin and the GPCRs SSTR3, Smoothened and GPR161 inside cilia. Strikingly, the single cell alga Chlamydomonas also requires its TOM1L2 orthologue to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

cell biology↗

bioRxiv2020

Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog; and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the putative phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

cell biology↗

bioRxiv2020

Nanobody-directed targeting of optogenetic tools reveals differential regulation of cilia length

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to temporarily manipulate and analyze ciliary signaling. Here, we describe a nanobodybased targeting approach for optogenetic tools that is applicable in vitro and in vivo and allows to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence. We functionally localized modifiers of cAMP signaling, i.e. the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, as well as the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

cell biology↗