The Experts below are selected from a list of 13665 Experts worldwide ranked by ideXlab platform
Esben Lorentzen - One of the best experts on this subject based on the ideXlab platform.
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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, Alethia Villasenor, Michael Heymann, Petra Schwille, Charlotte Kelley, 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. Cilia formation requires Intraflagellar transport (IFT) to move ciliary building blocks and signaling components into the cilium. Here authors use in vitro reconstitution and electron microscopy on IFT172 and reveal its ability to remodel large membrane surfaces into small vesicles.
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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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intraflagellar transport protein ift52 recruits ift46 to the basal body and flagella
Journal of Cell Science, 2017Co-Authors: Lei Wan, Esben Lorentzen, Michael Taschner, Xi Cheng, Kaiyao HuangAbstract:ABSTRACT Cilia are microtubule-based organelles and perform motile, sensing and signaling functions. The assembly and maintenance of cilia depend on intraflagellar transport (IFT). Besides ciliary localization, most IFT proteins accumulate at basal bodies. However, little is known about the molecular mechanism of basal body targeting of IFT proteins. We first identified the possible basal body-targeting sequence in IFT46 by expressing IFT46 truncation constructs in an ift46-1 mutant. The C-terminal sequence between residues 246–321, termed BBTS3, was sufficient to target YFP to basal bodies in the ift46-1 strain. Interestingly, BBTS3 is also responsible for the ciliary targeting of IFT46. BBTS3::YFP moves bidirectionally in flagella and interacts with other IFT complex B (IFT-B) proteins. Using IFT and motor mutants, we show that the basal body localization of IFT46 depends on IFT52, but not on IFT81, IFT88, IFT122, FLA10 or DHC1b. IFT52 interacts with IFT46 through residues L285 and L286 of IFT46 and recruits it to basal bodies. Ectopic expression of the C-terminal domain of IFT52 in the nucleus resulted in accumulation of IFT46 in nuclei. These data suggest that IFT52 and IFT46 can preassemble as a complex in the cytoplasm, which is then targeted to basal bodies.
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intraflagellar transport proteins 172 80 57 54 38 and 20 form a stable tubulin binding ift b2 complex
The EMBO Journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
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Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex
The EMBO journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
Michael Taschner - One of the best experts on this subject based on the ideXlab platform.
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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, Alethia Villasenor, Michael Heymann, Petra Schwille, Charlotte Kelley, 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. Cilia formation requires Intraflagellar transport (IFT) to move ciliary building blocks and signaling components into the cilium. Here authors use in vitro reconstitution and electron microscopy on IFT172 and reveal its ability to remodel large membrane surfaces into small vesicles.
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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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intraflagellar transport protein ift52 recruits ift46 to the basal body and flagella
Journal of Cell Science, 2017Co-Authors: Lei Wan, Esben Lorentzen, Michael Taschner, Xi Cheng, Kaiyao HuangAbstract:ABSTRACT Cilia are microtubule-based organelles and perform motile, sensing and signaling functions. The assembly and maintenance of cilia depend on intraflagellar transport (IFT). Besides ciliary localization, most IFT proteins accumulate at basal bodies. However, little is known about the molecular mechanism of basal body targeting of IFT proteins. We first identified the possible basal body-targeting sequence in IFT46 by expressing IFT46 truncation constructs in an ift46-1 mutant. The C-terminal sequence between residues 246–321, termed BBTS3, was sufficient to target YFP to basal bodies in the ift46-1 strain. Interestingly, BBTS3 is also responsible for the ciliary targeting of IFT46. BBTS3::YFP moves bidirectionally in flagella and interacts with other IFT complex B (IFT-B) proteins. Using IFT and motor mutants, we show that the basal body localization of IFT46 depends on IFT52, but not on IFT81, IFT88, IFT122, FLA10 or DHC1b. IFT52 interacts with IFT46 through residues L285 and L286 of IFT46 and recruits it to basal bodies. Ectopic expression of the C-terminal domain of IFT52 in the nucleus resulted in accumulation of IFT46 in nuclei. These data suggest that IFT52 and IFT46 can preassemble as a complex in the cytoplasm, which is then targeted to basal bodies.
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intraflagellar transport proteins 172 80 57 54 38 and 20 form a stable tubulin binding ift b2 complex
The EMBO Journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
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Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex
The EMBO journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
Brian D. Perkins - One of the best experts on this subject based on the ideXlab platform.
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Primary cilia are present on endothelial cells of the hyaloid vasculature but are not required for the development of the blood-retinal barrier.
PloS one, 2020Co-Authors: Lana M. Pollock, Brian D. Perkins, Bela Anand-apteAbstract:Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, IFT57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.
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early defects in photoreceptor outer segment morphogenesis in zebrafish IFT57 ift88 and ift172 intraflagellar transport mutants
Vision Research, 2009Co-Authors: Sujita Sukumaran, Brian D. PerkinsAbstract:Abstract Intraflagellar Transport (IFT) refers to a highly conserved process occurring in eukaryotic ciliated structures. In vertebrate photoreceptors, IFT mediates protein trafficking to the outer segments. The IFT particle is a multi-subunit complex and mutations in many individual components causes photoreceptor defects. In zebrafish, mutations in the IFT57 , ift88 , and ift172 genes result in retinal degeneration by 5 days post fertilization (dpf). Although the effects of these mutations on photoreceptor survival have been described, early developmental morphogenesis remains poorly understood. We used transmission electron microscopy and immunohistochemistry to examine these mutants at 60, 72, and 96 h post fertilization (hpf) and describe early photoreceptor morphogenesis defects.
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Zebrafish IFT57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect Hedgehog signaling
Developmental dynamics : an official publication of the American Association of Anatomists, 2009Co-Authors: Shannon Lunt, Tony E. Haynes, Brian D. PerkinsAbstract:Cilia formation requires intraflagellar transport (IFT) proteins. Recent studies indicate that mammalian Hedgehog (Hh) signaling requires cilia. It is unclear, however, if the requirement for cilia and IFT proteins in Hh signaling represents a general rule for all vertebrates. Here we examine zebrafish IFT57, ift88, and ift172 mutants and morphants for defects in Hh signaling. Although IFT57 and ift88 mutants and morphants contained residual maternal protein, the cilia were disrupted. In contrast to previous genetic studies in mouse, mutations in zebrafish IFT genes did not affect the expression of Hh target genes in the neural tube and forebrain and had no quantitative effect on Hh target gene expression. Zebrafish IFT mutants also exhibited no dramatic changes in the craniofacial skeleton, somite formation, or motor neuron patterning. Thus, our data indicate the requirement for cilia in the Hh signal transduction pathway may not represent a universal mechanism in vertebrates. Developmental Dynamics 238:1744–1759, 2009. © 2009 Wiley-Liss, Inc.
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the intraflagellar transport protein IFT57 is required for cilia maintenance and regulates ift particle kinesin ii dissociation in vertebrate photoreceptors
Development, 2008Co-Authors: Bryan L Krock, Brian D. PerkinsAbstract:1. 1. Krock B. L., 2. et al. 2008. J. Cell Sci. doi:10.1242/jcs.029397 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJournal%2Bof%2BCell%2BScience%26rft.stitle%253DJ.%2BCell%2BSci.%26rft.issn%253D0021-9533%26rft.aulast%253DKrock%26rft.
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the intraflagellar transport protein IFT57 is required for cilia maintenance and regulates ift particle kinesin ii dissociation in vertebrate photoreceptors
Journal of Cell Science, 2008Co-Authors: Bryan L Krock, Brian D. PerkinsAbstract:Defects in protein transport within vertebrate photoreceptors can result in photoreceptor degeneration. In developing and mature photoreceptors, proteins targeted to the outer segment are transported through the connecting cilium via the process of intraflagellar transport (IFT). In studies of vertebrate IFT, mutations in any component of the IFT particle typically abolish ciliogenesis, suggesting that IFT proteins are equally required for IFT. To determine whether photoreceptor outer segment formation depends equally on individual IFT proteins, we compared the retinal phenotypes of IFT57 and IFT88 mutant zebrafish. IFT88 mutants failed to form outer segments, whereas IFT57 mutants formed short outer segments with reduced amounts of opsin. Our phenotypic analysis revealed that IFT57 is not essential for IFT, but is required for efficient IFT. In co-immunoprecipitation experiments from whole-animal extracts, we determined that kinesin II remained associated with the IFT particle in the absence of IFT57, but IFT20 did not. Additionally, kinesin II did not exhibit ATP-dependent dissociation from the IFT particle in IFT57 mutants. We conclude that IFT20 requires IFT57 to associate with the IFT particle and that IFT57 and/or IFT20 mediate kinesin II dissociation.
Sagar Bhogaraju - One of the best experts on this subject based on the ideXlab platform.
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intraflagellar transport proteins 172 80 57 54 38 and 20 form a stable tubulin binding ift b2 complex
The EMBO Journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
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Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex
The EMBO journal, 2016Co-Authors: Michael Taschner, Sagar Bhogaraju, Kristina Weber, Andre Mourao, Melanie Vetter, Mayanka Awasthi, Marc Stiegler, Esben LorentzenAbstract:Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.
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biochemical mapping of interactions within the intraflagellar transport ift b core complex ift52 binds directly to four other ift b subunits
Journal of Biological Chemistry, 2011Co-Authors: Michael Taschner, Sagar Bhogaraju, Michaela Morawetz, Melanie Vetter, Esben LorentzenAbstract:Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.
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crystal structure of the intraflagellar transport complex 25 27
The EMBO Journal, 2011Co-Authors: Sagar Bhogaraju, Michael Taschner, Michaela Morawetz, Claire Basquin, Esben LorentzenAbstract:The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 A resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.
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Crystal structure of the intraflagellar transport complex 25/27
The EMBO journal, 2011Co-Authors: Sagar Bhogaraju, Michael Taschner, Michaela Morawetz, Claire Basquin, Esben LorentzenAbstract:The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 A resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.
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 protein 74 is essential for mouse spermatogenesis and male fertility by regulating axonemal microtubule assembly in mice
bioRxiv, 2018Co-Authors: Lin Shi, Shiyang Zhang, Gregory J. Pazour, Ting Zhou, Qian Huang, Ling Zhang, Rex A. Hess, Zhibing ZhangAbstract:IFT74 is a component of the core intraflagellar transport (IFT) 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 in male germ cells were infertile associated with low sperm counts and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axoneme 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 were significantly reduced in the mutant mice, with the exception of IFT20 and IFT25. The levels of ODF2 and SPAG16L proteins were also not changed. However, the processed AKAP4 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 highlights a potential genetic factor (IFT74) that contributes to human infertility in men.
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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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specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system
Journal of Cell Science, 2014Co-Authors: Francesca Finetti, Gregory J. Pazour, Laura Patrussi, Giulia Masi, Anna Onnis, Donatella Galgano, Orso Maria Lucherini, Cosima T BaldariAbstract:T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5+ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.
