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Esben Lorentzen - One of the best experts on this subject based on the ideXlab platform.
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Binding of IFT22 to the Intraflagellar Transport complex is essential for flagellum assembly
The EMBO journal, 2019Co-Authors: Stefanie Wachter, Cécile Fort, Philippe Bastin, Jérôme Basquin, Jamin Jung, Shahaan Shafiq, Esben LorentzenAbstract:Abstract Intraflagellar Transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non‐conserved G4/G5 motifs. We show that IFT22 specifically associates with G‐nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP‐binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22‐74/81 interactions. Structure‐based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP‐loading is not strictly required.
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trafficking of ciliary membrane proteins by the Intraflagellar Transport bbsome machinery
Essays in Biochemistry, 2018Co-Authors: Jenna L Wingfield, Karlferdinand Lechtreck, Esben LorentzenAbstract:Bardet–Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require Intraflagellar Transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to Transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the Transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.
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Membrane association and remodeling by Intraflagellar Transport protein IFT172.
Nature communications, 2018Co-Authors: Qianmin Wang, Esben Lorentzen, Michael Taschner, Kristina A. Ganzinger, Charlotte F. Kelley, Alethia Villasenor, Michael Heymann, Petra Schwille, Naoko MizunoAbstract:The cilium is an organelle used for motility and cellular signaling. Intraflagellar Transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. How IFT controls the movement of ciliary components is currently poorly understood. IFT172 is the largest IFT subunit essential for ciliogenesis. Due to its large size, the characterization of IFT172 has been challenging. Using giant unilamellar vesicles (GUVs), we show that IFT172 is a membrane-interacting protein with the ability to remodel large membranes into small vesicles. Purified IFT172 has an architecture of two globular domains with a long rod-like protrusion, resembling the domain organization of coatomer proteins such as COPI-II or clathrin. IFT172 adopts two different conformations that can be manipulated by lipids or detergents: 1) an extended elongated conformation and 2) a globular closed architecture. Interestingly, the association of IFT172 with membranes is mutually exclusive with IFT57, implicating multiple functions for IFT172 within IFT.
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Structural basis of outer dynein arm Intraflagellar Transport by the Transport adaptor protein ODA16 and the Intraflagellar Transport protein IFT46.
The Journal of biological chemistry, 2017Co-Authors: Michael Taschner, André Mourão, Mayanka Awasthi, Jérôme Basquin, Esben LorentzenAbstract:Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The Transport of ODAs into cilia has been previously shown to require the Transport adaptor ODA16, as well as the Intraflagellar Transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and Transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.
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The Intraflagellar Transport Machinery
Cold Spring Harbor perspectives in biology, 2016Co-Authors: Michael Taschner, Esben LorentzenAbstract:Eukaryotic cilia and flagella are evolutionarily conserved organelles that protrude from the cell surface. The unique location and properties of cilia allow them to function in vital processes such as motility and signaling. Ciliary assembly and maintenance rely on Intraflagellar Transport (IFT), the bidirectional movement of a multicomponent Transport system between the ciliary base and tip. Since its initial discovery more than two decades ago, considerable effort has been invested in dissecting the molecular mechanisms of IFT in a variety of model organisms. Importantly, IFT was shown to be essential for mammalian development, and defects in this process cause a number of human pathologies known as ciliopathies. Here, we review current knowledge of IFT with a particular emphasis on the IFT machinery and specific mechanisms of ciliary cargo recognition and Transport.
Jonathan M. Scholey - One of the best experts on this subject based on the ideXlab platform.
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Illuminating the Intraflagellar Transport Machinery of Caenorhabditis Elegans
Biophysical Journal, 2012Co-Authors: Bram Prevo, Jonathan M. Scholey, Pierre Mangeol, Erwin J.g. PetermanAbstract:Molecular motors of the kinesin and dynein superfamilies are the driving force behind intracellular Transport, cell division and cell propulsion. Inside the chemosensory cilia of Caenorhabditis elegans two kinesin-2-family motors, kinesin-II and OSM-3-kinesin, cooperate to build and maintain the cilium, in a process called Intraflagellar Transport (IFT). In order to quantitatively asses IFT-kinesin function at endogenous expression levels, we have generated transgenic worms using Mos1-mediated single-copy integration of transgenes encoding fluorescently-labeled-IFT kinesins. Ultrasensitive wide-field and confocal fluorescence microscopy allows accurate mapping, counting, tracking and correlation of these molecular machines inside living, multicellular organisms. This approach allows unprecedented insight into IFT and motor-driven processes in general.
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Intraflagellar Transport delivers tubulin isotypes to sensory cilium middle and distal segments
Nature cell biology, 2011Co-Authors: Limin Hao, Ingrid Brust-mascher, Bram Prevo, Melanie Thein, Gul Civelekoglu-scholey, Seyda Acar, Shai Shaham, Jonathan M. ScholeyAbstract:Sensory cilia are assembled and maintained by kinesin-2-dependent Intraflagellar Transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual Transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length.
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Analysis of Intraflagellar Transport in C. elegans sensory cilia.
Methods in cell biology, 2009Co-Authors: Limin Hao, Seyda Acar, James E. Evans, Jonathan M. ScholeyAbstract:Cilia are assembled and maintained by Intraflagellar Transport (IFT), the motor-dependent, bidirectional movement of multiprotein complexes, called IFT particles, along the axoneme. The sensory cilia of Caenorhabditis elegans represent very useful objects for studying IFT because of the availability of in vivo time-lapse fluorescence microscopy assays of IFT and multiple ciliary mutants. In this system there are 60 sensory neurons, each having sensory cilia on the endings of their dendrites, and most components of the IFT machinery operating in these structures have been identified using forward and reverse genetic approaches. By analyzing the rate of IFT along cilia within living wild-type and mutant animals, two anterograde and one retrograde IFT motors were identified, the functional coordination of the two anterograde kinesin-2 motors was established and the Transport properties of all the known IFT particle components have been characterized. The anterograde kinesin motors have been heterologously expressed and purified, and their biochemical properties have been characterized using MT gliding and single molecule motility assays. In this chapter, we summarize how the tools of genetics, cell biology, electron microscopy, and biochemistry are being used to dissect the composition and mechanism of action of IFT motors and IFT particles in C. elegans.
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Intraflagellar Transport at a glance.
Journal of Cell Science, 2009Co-Authors: Limin Hao, Jonathan M. ScholeyAbstract:Intraflagellar Transport (IFT) is the bidirectional Transport of multisubunit protein complexes, called IFT particles, along axonemal microtubules (MTs) beneath the ciliary membrane. IFT plays essential roles in the assembly and function of cilia and flagella by contributing to cell motility,
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Intraflagellar Transport motors in cilia: Moving along the cell's antenna
Journal of Cell Biology, 2008Co-Authors: Jonathan M. ScholeyAbstract:Intraflagellar Transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo Transport. I review evidence that kinesin-2 motors physically Transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.
Joel L. Rosenbaum - One of the best experts on this subject based on the ideXlab platform.
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Isolation of Intraflagellar Transport trains
Cytoskeleton (Hoboken N.J.), 2013Co-Authors: Caterina Mencarelli, Joel L. Rosenbaum, Aaron P. Mitchell, Roberto Leoncini, Pietro LupettiAbstract:The Intraflagellar Transport (IFT) system was first identified in situ by electron microscopy in thin sections of plastic-embedded flagella as linear arrays of electrondense particles, located between the B tubules of the outer doublets and the flagellar membrane. These arrays of particles are referred to as IFT trains. Upon membrane rupture, IFT trains are thought to easily dissociate to yield soluble IFT particles, which are commonly purified through sucrose gradients as ∼16-17S complexes. The latters easily dissociate into two subcomplexes, named A and B. We report here the isolation, visualization, and identification by immunolabeling of flexible strings of IFT particles, which are structurally similar to in situ IFT trains and appear to be formed by both complex A and complex B polypeptides. Moreover, the particles forming isolated IFT trains are structurally similar to the individual particles found in the ∼17S gradient peak. Our results provide the first direct evidence that ∼17S particles do indeed compose the IFT trains. The paper also represents the first isolation of the IFT trains, and opens new possibilities for higher resolution studies on their structure and how particles are attached to each other to form the particle trains.
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Intraflagellar Transport: a new player at the immune synapse.
Trends in immunology, 2011Co-Authors: Francesca Finetti, Joel L. Rosenbaum, Silvia Rossi Paccani, Cosima T. BaldariAbstract:The assembly and maintenance of primary cilia, which orchestrate signaling pathways centrally implicated in cell proliferation, differentiation and migration, are ensured by multimeric protein particles in a process known as Intraflagellar Transport (IFT). It has recently been demonstrated that a number of IFT components are expressed in hematopoietic cells, which have no cilia. Here, we summarize data for an unexpected role of IFT proteins in immune synapse assembly and intracellular membrane trafficking in T lymphocytes, and discuss the hypothesis that the immune synapse could represent the functional homolog of the primary cilium in these cells.
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Intraflagellar Transport: it's not just for cilia anymore.
Current opinion in cell biology, 2009Co-Authors: Cosima T. Baldari, Joel L. RosenbaumAbstract:Recently published information on the role of Intraflagellar Transport (IFT) polypeptides in vesicle exocytosis is reviewed, describing the formation of the immune synapse in nonciliated cells as an example. A hypothesis is detailed suggesting that all polypeptides which enter the cilium, both membrane and axonemal, do so in association, first, with cytoplasmic vesicles which exocytose adjacent to the ciliary basal body, and then with the ciliary membrane. Axonemal proteins are moved to the ciliary tip by peripheral association with the inner aspects of the ciliary membrane by cannonical ciliary IFT. At the tip, some polypeptides are released for axonemal assembly, and others are budded off as part of vesicular exosomes into the environment. It is proposed that the cilium, in addition to being a sensory and motile organelle, is also a secretory organelle.
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Intraflagellar Transport is required for polarized recycling of the tcr cd3 complex to the immune synapse
Nature Cell Biology, 2009Co-Authors: Francesca Finetti, Gregory J. Pazour, Joel L. Rosenbaum, Silvia Rossi Paccani, Maria Giovanna Riparbelli, Emiliana Giacomello, Giuseppe Perinetti, Cosima T. BaldariAbstract:IFT20, known to control Intraflagellar Transport during cilia biogenesis, is also expressed in lymphoid and myeloid non-ciliated cells. IFT20 is localized to the secretory pathway in T-lymphocytes and translocates to the immune synapse on antigen engagement to modulate the recycling of T-cell receptors.
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Intraflagellar Transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse.
Nature cell biology, 2009Co-Authors: Francesca Finetti, Gregory J. Pazour, Joel L. Rosenbaum, Silvia Rossi Paccani, Maria Giovanna Riparbelli, Emiliana Giacomello, Giuseppe Perinetti, Cosima T. BaldariAbstract:IFT20, known to control Intraflagellar Transport during cilia biogenesis, is also expressed in lymphoid and myeloid non-ciliated cells. IFT20 is localized to the secretory pathway in T-lymphocytes and translocates to the immune synapse on antigen engagement to modulate the recycling of T-cell receptors.
Gregory J. Pazour - One of the best experts on this subject based on the ideXlab platform.
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Ubiquitin Links Smoothened to Intraflagellar Transport to Regulate Hedgehog Signaling
2019Co-Authors: Paurav B. Desai, Michael W. Stuck, Gregory J. PazourAbstract:In the absence of hedgehog ligand, patched-1 (Ptch1) localizes to cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon ligand binding, Ptch1 is removed from cilia, Smo is derepressed and accumulates in cilia where it activates signaling. The mechanisms regulating these dynamic movements are not well understood but defects in Intraflagellar Transport components including Ift27 and the BBSome cause Smo to accumulate in cilia without pathway activation. We find that in the absence of ligand-induced pathway activation, Smo is ubiquitinated and removed from cilia, and this process is dependent on Ift27 and BBSome components. Activation of hedgehog signaling decreases Smo ubiquitination, and ciliary removal, resulting in its accumulation. Blocking ubiquitination of Smo by an E1 ligase inhibitor or by mutating two lysine residues in intracellular loop three cause Smo to aberrantly accumulate in cilia without pathway activation. These data provide a mechanism to control Smo9s ciliary level during hedgehog signaling by regulating the ubiquitination state of the receptor.
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Intraflagellar Transport protein 74 is essential for spermatogenesis and male fertility in mice
Biology of Reproduction, 2019Co-Authors: Lin Shi, Shiyang Zhang, Gregory J. Pazour, Ting Zhou, Qian Huang, Ling Zhang, Rex A. Hess, Zhibing ZhangAbstract:Intraflagellar Transport protein 74 (IFT74) is a component of the core Intraflagellar Transport complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axonemes in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 and sperm-associated antigen 16L proteins were also not changed. However, the processed A-Kinase anchor protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.
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Intraflagellar Transport is deeply integrated in hedgehog signaling.
Molecular biology of the cell, 2018Co-Authors: Thibaut Eguether, Fabrice P. Cordelières, Gregory J. PazourAbstract:The vertebrate hedgehog pathway is organized in primary cilia, and hedgehog components relocate into or out of cilia during signaling. Defects in Intraflagellar Transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining whether IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similarly to other IFTs, allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway in Ift27 mutant cells showed that IFT is extensively involved in the pathway. Similar analysis of Bbs mutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to sequester IFT-B proteins at the mitochondria in cells with fully formed cilia did not affect the delivery of Smo to cilia, suggesting that this membrane protein may not require IFT-B for delivery.
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IFT25 Links the Signal-Dependent Movement of Hedgehog Components to Intraflagellar Transport
Developmental cell, 2012Co-Authors: Brian T. Keady, Rajeev Samtani, Kimimasa Tobita, Maiko Tsuchya, Jovenal T. San Agustin, John A. Follit, Julie A. Jonassen, Ramiah Subramanian, Gregory J. PazourAbstract:The Intraflagellar Transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent Transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.
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Intraflagellar Transport is required for polarized recycling of the tcr cd3 complex to the immune synapse
Nature Cell Biology, 2009Co-Authors: Francesca Finetti, Gregory J. Pazour, Joel L. Rosenbaum, Silvia Rossi Paccani, Maria Giovanna Riparbelli, Emiliana Giacomello, Giuseppe Perinetti, Cosima T. BaldariAbstract:IFT20, known to control Intraflagellar Transport during cilia biogenesis, is also expressed in lymphoid and myeloid non-ciliated cells. IFT20 is localized to the secretory pathway in T-lymphocytes and translocates to the immune synapse on antigen engagement to modulate the recycling of T-cell receptors.
Cosima T. Baldari - One of the best experts on this subject based on the ideXlab platform.
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Intraflagellar Transport: a new player at the immune synapse.
Trends in immunology, 2011Co-Authors: Francesca Finetti, Joel L. Rosenbaum, Silvia Rossi Paccani, Cosima T. BaldariAbstract:The assembly and maintenance of primary cilia, which orchestrate signaling pathways centrally implicated in cell proliferation, differentiation and migration, are ensured by multimeric protein particles in a process known as Intraflagellar Transport (IFT). It has recently been demonstrated that a number of IFT components are expressed in hematopoietic cells, which have no cilia. Here, we summarize data for an unexpected role of IFT proteins in immune synapse assembly and intracellular membrane trafficking in T lymphocytes, and discuss the hypothesis that the immune synapse could represent the functional homolog of the primary cilium in these cells.
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Intraflagellar Transport: it's not just for cilia anymore.
Current opinion in cell biology, 2009Co-Authors: Cosima T. Baldari, Joel L. RosenbaumAbstract:Recently published information on the role of Intraflagellar Transport (IFT) polypeptides in vesicle exocytosis is reviewed, describing the formation of the immune synapse in nonciliated cells as an example. A hypothesis is detailed suggesting that all polypeptides which enter the cilium, both membrane and axonemal, do so in association, first, with cytoplasmic vesicles which exocytose adjacent to the ciliary basal body, and then with the ciliary membrane. Axonemal proteins are moved to the ciliary tip by peripheral association with the inner aspects of the ciliary membrane by cannonical ciliary IFT. At the tip, some polypeptides are released for axonemal assembly, and others are budded off as part of vesicular exosomes into the environment. It is proposed that the cilium, in addition to being a sensory and motile organelle, is also a secretory organelle.
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Intraflagellar Transport is required for polarized recycling of the tcr cd3 complex to the immune synapse
Nature Cell Biology, 2009Co-Authors: Francesca Finetti, Gregory J. Pazour, Joel L. Rosenbaum, Silvia Rossi Paccani, Maria Giovanna Riparbelli, Emiliana Giacomello, Giuseppe Perinetti, Cosima T. BaldariAbstract:IFT20, known to control Intraflagellar Transport during cilia biogenesis, is also expressed in lymphoid and myeloid non-ciliated cells. IFT20 is localized to the secretory pathway in T-lymphocytes and translocates to the immune synapse on antigen engagement to modulate the recycling of T-cell receptors.
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Intraflagellar Transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse.
Nature cell biology, 2009Co-Authors: Francesca Finetti, Gregory J. Pazour, Joel L. Rosenbaum, Silvia Rossi Paccani, Maria Giovanna Riparbelli, Emiliana Giacomello, Giuseppe Perinetti, Cosima T. BaldariAbstract:IFT20, known to control Intraflagellar Transport during cilia biogenesis, is also expressed in lymphoid and myeloid non-ciliated cells. IFT20 is localized to the secretory pathway in T-lymphocytes and translocates to the immune synapse on antigen engagement to modulate the recycling of T-cell receptors.