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.

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.

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.

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.

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.

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.

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.

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.

Tailored Educational Approaches for Consumer Health: A Model to Address Health Promotion in an Era of Personalized Medicine.

Purpose: To develop a model, based on market segmentation, to improve the quality and efficiency of health promotion materials and programs.

Design: Market segmentation to create segments (groups) based on a cross-sectional questionnaire measuring individual characteristics and preferences for health information. Educational and delivery recommendations developed for each group.

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.

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 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.

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.

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.

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.

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.

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.

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)).

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.

Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly.

Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially-encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome.

Tailored Educational Approaches for Consumer Health (TEACH): a model system for addressing health communication.

The Consumer Health Education Institute (CHEDI) has developed a model system to improve the quality and effectiveness of patient education and health communication. Through assessment of characteristics and preferences, segmentation into groups and matching with the appropriate materials, we have demonstrated that patients and health consumers have different health information needs and preferences which show promise as a basis for selecting or designing the most appropriate materials or programs.

A role for Tim21 in membrane-potential-dependent preprotein sorting in mitochondria.

The mitochondrial inner membrane harbors complexes of the respiratory chain and translocase complexes for preproteins. The membrane potential generated by the respiratory chain is essential for ATP production by the mitochondrial ATP synthase and as a driving force for protein import. It is generally believed that the preprotein translocases just use the membrane potential without getting into physical contact with respiratory-chain complexes.

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello.