Emerging principles of primary cilia dynamics in controlling tissue organization and function.

Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.

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 (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 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 target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and 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. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

Time-resolved proteomics profiling of the ciliary Hedgehog response.

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 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.

Phosphorylation and Ubiquitylation Regulate Protein Trafficking, Signaling, and the Biogenesis of Primary Cilia.

The primary cilium is a solitary, microtubule-based membrane protrusion extending from the surface of quiescent cells that senses the cellular environment and triggers specific cellular responses. The functions of primary cilia require not only numerous different components but also their regulated interplay. The cilium performs highly dynamic processes, such as cell cycle-dependent assembly and disassembly as well as delivery, modification, and removal of signaling components to perceive and process external signals. On a molecular level, these processes often rely on a stringent control of key modulatory proteins, of which the activity, localization, and stability are regulated by post-translational modifications (PTMs). While an increasing number of PTMs on ciliary components are being revealed, our knowledge on the identity of the modifying enzymes and their modulation is still limited. Here, we highlight recent findings on cilia-specific phosphorylation and ubiquitylation events. Shedding new light onto the molecular mechanisms that regulate the sensitive equilibrium required to maintain and remodel primary cilia functions, we discuss their implications for cilia biogenesis, protein trafficking, and cilia signaling processes.

Signal transduction in primary cilia - analyzing and manipulating GPCR and second messenger signaling.

The primary cilium projects from the surface of most vertebrate cells, where it senses extracellular signals to regulate diverse cellular processes during tissue development and homeostasis. Dysfunction of primary cilia underlies the pathogenesis of severe diseases, commonly referred to as ciliopathies. Primary cilia contain a unique protein repertoire that is distinct from the cell body and the plasma membrane, enabling the spatially controlled transduction of extracellular cues. G-protein coupled receptors (GPCRs) are key in sensing environmental stimuli that are transmitted via second messenger signaling into a cellular response. Here, we will give an overview of the role of GPCR signaling in primary cilia, and how ciliary GPCR signaling can be targeted by pharmacology, chemogenetics, and optogenetics.

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium.

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 has been challenging due to the lack of tools to investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylyl cyclase bPAC and the light-activated phosphodiesterase LAPD, and 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.

Establishing Cell Culture-Based Experimental Setups for Proximity Labeling Using Ascorbate Peroxidase (APEX).

Proximity labeling by ascorbate peroxidase (APEX) requires appropriate experimental setups that generate sufficient signal over background as a prerequisite for downstream analyses by mass spectrometry. Cell culture-based systems are easily accessible, yet, for proximity labeling of small structures must be carefully optimized in order to give satisfying results. How to establish and characterize APEX cell lines will be the topic of this chapter.

Establishing and regulating the composition of cilia for signal transduction.

The primary cilium is a hair-like surface-exposed organelle of the eukaryotic cell that decodes a variety of signals - such as odorants, light and Hedgehog morphogens - by altering the local concentrations and activities of signalling proteins. Signalling within the cilium is conveyed through a diverse array of second messengers, including conventional signalling molecules (such as cAMP) and some unusual intermediates (such as sterols). Diffusion barriers at the ciliary base establish the unique composition of this signalling compartment, and cilia adapt their proteome to signalling demands through regulated protein trafficking. Much progress has been made on the molecular understanding of regulated ciliary trafficking, which encompasses not only exchanges between the cilium and the rest of the cell but also the shedding of signalling factors into extracellular vesicles.

Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

Proteomics of Primary Cilia by Proximity Labeling.

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that ß-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

The heme a synthase Cox15 associates with cytochrome c oxidase assembly intermediates during Cox1 maturation.

Cox1, the core subunit of the cytochrome c oxidase, receives two heme a cofactors during assembly of the 13-subunit enzyme complex. However, at which step of the assembly process and how heme is inserted into Cox1 have remained an enigma. Shy1, the yeast SURF1 homolog, has been implicated in heme transfer to Cox1, whereas the heme a synthase, Cox15, catalyzes the final step of heme a synthesis. Here we performed a comprehensive analysis of cytochrome c oxidase assembly intermediates containing Shy1. Our analyses suggest that Cox15 displays a role in cytochrome c oxidase assembly, which is independent of its functions as the heme a synthase. Cox15 forms protein complexes with Shy1 and also associates with Cox1-containing complexes independently of Shy1 function. These findings indicate that Shy1 does not serve as a mobile heme carrier between the heme a synthase and maturing Cox1 but rather cooperates with Cox15 for heme transfer and insertion in early assembly intermediates of cytochrome c oxidase.

MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation.

Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.

Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane.

Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA-specific activator proteins and occurs on membrane-associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K(+)/H(+) or Ca(2+)/H(+) antiporters in the inner membrane. Here, we identify the conserved ribosome-binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38(RBD) displays a 14-3-3-like fold despite any similarity to 14-3-3-proteins at the primary sequence level and thus represents the first 14-3-3-like protein in mitochondria. The 14-3-3-like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.

Mimicking a SURF1 allele reveals uncoupling of cytochrome c oxidase assembly from translational regulation in yeast.

Defects in mitochondrial energy metabolism lead to severe human disorders, mainly affecting tissues especially dependent on oxidative phosphorylation, such as muscle and brain. Leigh Syndrome describes a severe encephalomyopathy in infancy, frequently caused by mutations in SURF1. SURF1, termed Shy1 in Saccharomyces cerevisiae, is a conserved assembly factor for the terminal enzyme of the respiratory chain, cytochrome c oxidase. Although the molecular function of SURF1/Shy1 is still enigmatic, loss of function leads to cytochrome c oxidase deficiency and reduced expression of the central subunit Cox1 in yeast. Here, we provide insights into the molecular mechanisms leading to disease through missense mutations in codons of the most conserved amino acids in SURF1. Mutations affecting G(124) do not compromise import of the SURF1 precursor protein but lead to fast turnover of the mature protein within the mitochondria. Interestingly, an Y(274)D exchange neither affects stability nor localization of the protein. Instead, SURF1(Y274D) accumulates in a 200 kDa cytochrome c oxidase assembly intermediate. Using yeast as a model, we demonstrate that the corresponding Shy1(Y344D) is able to overcome the stage where cytochrome c oxidase assembly links to the feedback regulation of mitochondrial Cox1 expression. However, Shy1(Y344D) impairs the assembly at later steps, most apparent at low temperature and exhibits a dominant-negative phenotype upon overexpression. Thus, exchanging the conserved tyrosine (Y(344)) with aspartate in yeast uncouples translational regulation of Cox1 from cytochrome c oxidase assembly and provides evidence for the dual functionality of Shy1.

Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation.

Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.

Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria.

Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.

Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation.

Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.

Distinct forms of mitochondrial TOM-TIM supercomplexes define signal-dependent states of preprotein sorting.

Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23(SORT)). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23(SORT) and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.

Protein transport machineries for precursor translocation across the inner mitochondrial membrane.

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.