Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after ...stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.
Cilia are surface-exposed organelles that dynamically concentrate signaling molecules to organize sensory, developmental and homeostatic pathways. Entry and exit of signaling receptors is germane to ...the processing of signals and the molecular machines for entry and exit have started to emerge. The IFT-A complex and its membrane recruitment factor Tulp3 complex promotes the entry of signaling receptors into cilia while the BBSome and its membrane recruitment factor Arl6GTP ferry activated signaling receptors out of cilia. Ciliary exit is a surprisingly complex process entailing passage through a first diffusion barrier at the transition zone, diffusion inside an intermediate compartment and crossing of a periciliary diffusion barrier. The two barriers may organize a privileged compartment where activated signaling receptors transiently reside.
How do cilia organize signalling cascades? Nachury, Maxence V.
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
09/2014, Volume:
369, Issue:
1650
Journal Article
Peer reviewed
Open access
Cilia and flagella are closely related centriole-nucleated protrusions of the cell with roles in motility and signal transduction. Two of the best-studied signalling pathways organized by cilia are ...the transduction cascade for the morphogen Hedgehog in vertebrates and the mating pathway that initiates gamete fusion in the unicellular green alga Chlamydomonas reinhardtii. What is the role of cilia in these signalling transduction cascades? In both Hedgehog and mating pathways, all signalling intermediates have been found to localize to cilia, and, for some signalling factors, ciliary localization is regulated by pathway activation. Given a concentration factor of three orders of magnitude provided by translocating a protein into the cilium, the compartment model proposes that cilia act as miniaturized reaction tubes bringing signalling factors and processing enzymes in close proximity. On the other hand, the scaffolding model views the intraflagellar transport machinery, whose primary function is to build cilia and flagella, as a molecular scaffold for the mating transduction cascade at the flagellar membrane. While these models may coexist, it is hoped that a precise understanding of the mechanisms that govern signalling inside cilia will provide a satisfying answer to the question ‘how do cilia organize signalling?’. This review covers the evidence supporting each model of signalling and outlines future directions that may address which model applies in given biological settings.
A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small guanosine triphosphatase Arl6, which triggers BBSome coat ...polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. In this study, we found that activation of the ciliary G protein-coupled receptors (GPCRs) Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive, and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. However, the removal of activated GPCRs from cilia was inefficient because a second periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first move activated GPCRs through the transition zone before a periciliary barrier can be crossed.
Regulated trafficking of G protein-coupled receptors (GPCRs) controls cilium-based signaling pathways. β-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet-Biedl ...syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a β-arrestin-dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.
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 β-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.
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•APEX labeling enables proteomic analyses of a non-membrane-enclosed compartment•Cilia-APEX identifies the kinases PKA, AMPK, and LKB1 in primary cilia•PKA functions inside cilia to phosphorylate GLI3 and regulate Hedgehog signaling•Proteomic profiling of Ift27/Bbs19 cilia detects ciliary accumulation of BBSome
Primary cilia organize cellular signaling events in a specialized microenvironment. Mick et al. apply proximity labeling using cilia-APEX to study the ciliary proteome. They uncover unexpected signaling molecules, including kinases PKA and AMPK, inside cilia and further use a proteomic profiling approach to unravel molecular defects of Ift27/Bbs19 mutant cilia.
The assembly and signaling properties of cilia rely on intraflagellar transport (IFT) trains moving proteins into, within, and out of cilia. A flurry of near-atomic models of the multiprotein ...complexes that make up IFT trains has revealed new conformational changes, which may underlie the switch between anterograde and retrograde intraflagellar transport.
The assembly and signaling properties of cilia rely on intraflagellar transport (IFT) trains moving proteins into, within, and out of cilia. A flurry of near-atomic models of the multiprotein complexes that make up IFT trains has revealed new conformational changes, which may underlie the switch between anterograde and retrograde intraflagellar transport.
Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule ...stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that ...microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.
Cilia sense and transduce sensory stimuli, homeostatic cues and developmental signals by orchestrating signaling reactions. Extracellular vesicles (EVs) that bud from the ciliary membrane have ...well-studied roles in the disposal of excess ciliary material, most dramatically exemplified by the shedding of micrometer-sized blocks by photoreceptors. Shedding of EVs by cilia also affords cells with a powerful means to shorten cilia. Finally, cilium-derived EVs may enable cell-cell communication in a variety of organisms, ranging from single-cell parasites and algae to nematodes and vertebrates. Mechanistic understanding of EV shedding by cilia is an active area of study, and future progress may open the door to testing the function of ciliary EV shedding in physiological contexts. In this Cell Science at a Glance and the accompanying poster, we discuss the molecular mechanisms that drive the shedding of ciliary material into the extracellular space, the consequences of shedding for the donor cell and the possible roles that ciliary EVs may have in cell non-autonomous contexts.
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