Genetic interaction of mammalian IFT-A paralogs regulates cilia disassembly, ciliary protein trafficking, Hedgehog signaling and embryogenesis

AbstractPrimary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bi-directional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues of Chlamydomonas IFT-A gene, IFT139, namely Thm1 (also known as Ttc21b) and Thm2 (Ttc21a). Thm1 loss causes perinatal lethality, and Thm2 loss allows survival into adulthood. At E14.5, the number of Thm1;Thm2 double mutant embryos is lower than that for a Mendelian ratio, indicating deletion of Thm1 and Thm2 causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF. Thm1-mutant MEF show decreased cilia assembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins. Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG. Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of a Thm1 allele impairs response to SAG. Further, Thm1;Thm2 double mutant MEF show enhanced cilia disassembly, and relative to Thm1-null MEF, increased impairment of IFT81 retrograde transport and of INPP5E ciliary import. Thus, Thm1 and Thm2 have unique and redundant roles in MEF. Thm1 regulates cilia assembly, and together with Thm2, cilia disassembly. Moreover, Thm1 alone and together with Thm2, regulates ciliary protein trafficking, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlying Thm1-, Thm2- or IFT-A-mediated ciliopathies.

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CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

PLoS ONE ◽

10.1371/journal.pone.0258497 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258497

Author(s):

Tatsuro Noguchi ◽

Kentaro Nakamura ◽

Yuuki Satoda ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

Extracellular Vesicles ◽

Protein Trafficking ◽

Hedgehog Signaling ◽

Intraflagellar Transport ◽

Wild Type ◽

Ciliary Membrane ◽

Ciliary Protein ◽

Exogenous Expression ◽

Turnaround Process

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

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Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis

10.1101/2020.01.26.920165 ◽

2020 ◽

Author(s):

Bailey A Allard ◽

Wei Wang ◽

Tana S Pottorf ◽

Hammad Mumtaz ◽

Luciane M Silva ◽

...

Keyword(s):

Hedgehog Signaling ◽

Primary Cilia ◽

Genetic Interaction ◽

Skeletal Development ◽

Intraflagellar Transport ◽

Genetic Syndromes ◽

Mineral Density ◽

Proliferation Zone ◽

Trabecular Bone Mineral Density ◽

Redundant Functions

AbstractCiliopathies are genetic syndromes that link osteochondrodysplasias to dysfunction of primary cilia. Primary cilia extend from the surface of bone and cartilage cells, to receive extracellular cues and mediate signaling pathways. Mutations in several genes that encode components of the intraflagellar transport-A ciliary protein complex have been identified in skeletal ciliopathies, including THM1. Here, we report a role for genetic interaction between Thm1 and its paralog, Thm2, in skeletogenesis. THM2 localizes to the ciliary axoneme, but unlike its paralog, Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. Since paralogs often have redundant functions, we crossed a Thm1 null (aln) allele into the Thm2 colony. After 5 generations of backcrossing the colony onto a C57BL6/J background, we observed that by postnatal day 14, Thm2-/-; Thm1aln/+ mice are smaller than control littermates. Thm2-/-; Thm1aln/+ mice exhibit shortened long bones, narrow ribcage, shortened cranium and mandibular defects. Mutant mice also show aberrant architecture of the tibial growth plate, with an expanded proliferation zone and diminished hypertrophic zone, indicating impaired chondrocyte differentiation. Using microcomputed tomography, Thm2-/-; Thm1aln/+ tibia were revealed to have reduced cortical and trabecular bone mineral density. Deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog (Hh) pathway, exacerbated the small phenotype of Thm2-/-; Thm1aln/+ mice and caused small stature in Thm2-null mice. Together, these data reveal Thm2 as a novel locus that sensitizes to Hh signaling in skeletal development. Further, Thm2-/-; Thm1aln/+ mice present a new postnatal ciliopathy model of osteochondrodysplasia.

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ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins

Scientific Reports ◽

10.1038/s41598-019-52830-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Mona Alsolami ◽

Stefanie Kuhns ◽

Manal Alsulami ◽

Oliver E. Blacque

Keyword(s):

Sonic Hedgehog ◽

Primary Cilium ◽

Molecular Motors ◽

Hedgehog Signaling ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Signaling Proteins ◽

Sonic Hedgehog Signaling ◽

And Function

Abstract Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

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Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia

Human Molecular Genetics ◽

10.1093/hmg/ddab034 ◽

2021 ◽

Author(s):

Yamato Ishida ◽

Takuya Kobayashi ◽

Shuhei Chiba ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

Protein Trafficking ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Missense Variant ◽

Cellular Level ◽

Compound Heterozygous ◽

Hypomorphic Allele ◽

Genes Encoding ◽

Specific Proteins ◽

Compound Heterozygous Mutations

Abstract Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140, and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

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The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Molecular Biology of the Cell ◽

10.1091/mbc.e14-05-0961 ◽

2014 ◽

Vol 25 (17) ◽

pp. 2620-2633 ◽

Cited By ~ 33

Author(s):

Thierry Blisnick ◽

Johanna Buisson ◽

Sabrina Absalon ◽

Alexandra Marie ◽

Nadège Cayet ◽

...

Keyword(s):

Protein Complexes ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

High Concentration ◽

Anterograde Transport ◽

Heavy Chains ◽

Cilia And Flagella ◽

The Stability ◽

Intermediate Chains ◽

Dynein Heavy Chains

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.

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Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0654 ◽

2019 ◽

Vol 30 (7) ◽

pp. 828-837 ◽

Cited By ~ 5

Author(s):

T. Tony Yang ◽

Minh Nguyet Thi Tran ◽

Weng Man Chong ◽

Chia-En Huang ◽

Jung-Chi Liao

Keyword(s):

Particle Tracking ◽

Single Particle ◽

Primary Cilia ◽

Protein Complexes ◽

Retinal Pigment ◽

Intraflagellar Transport ◽

Vital Role ◽

Single Particle Tracking ◽

Retinal Pigment Epithelial Cells ◽

Localization Microscopy

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins “switched gears” multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

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ARL13B regulates Sonic Hedgehog signaling from outside primary cilia

10.1101/711671 ◽

2019 ◽

Author(s):

Eduardo D. Gigante ◽

Megan R. Taylor ◽

Anna A. Ivanova ◽

Richard A. Kahn ◽

Tamara Caspary

Keyword(s):

Signal Transduction ◽

Sonic Hedgehog ◽

Protein Trafficking ◽

Hedgehog Signaling ◽

Primary Cilia ◽

Complete Loss ◽

Wild Type ◽

Sonic Hedgehog Signaling ◽

Biochemical Function

AbstractARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

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ARL13B regulates Sonic hedgehog signaling from outside primary cilia

eLife ◽

10.7554/elife.50434 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 6

Author(s):

Eduardo D Gigante ◽

Megan R Taylor ◽

Anna A Ivanova ◽

Richard A Kahn ◽

Tamara Caspary

Keyword(s):

Signal Transduction ◽

Sonic Hedgehog ◽

Protein Trafficking ◽

Hedgehog Signaling ◽

Primary Cilia ◽

Complete Loss ◽

Wild Type ◽

Sonic Hedgehog Signaling ◽

Biochemical Function

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

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IFT20 is critical for early chondrogenesis during endochondral ossification

10.1101/2021.06.25.449945 ◽

2021 ◽

Author(s):

Hiroyuki Yamaguchi ◽

Megumi Kitami ◽

Karin H Uchima Koecklin ◽

Li He ◽

Jianbo Wang ◽

...

Keyword(s):

Hedgehog Signaling ◽

Endochondral Ossification ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Sox9 Expression ◽

Skeletal Defects ◽

The Family ◽

Limb Outgrowth ◽

Mutant Cells ◽

Critical Process

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, it remains unclear how each IFT protein performs its unique function to regulate endochondral ossification. Here, we show that intraflagellar transport 20 (IFT20) is required for early chondrogenesis. Utilizing three osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre, Col2-Cre and Aggrecan-CreERT2), we deleted Ift20 to examine its function. While chondrocyte-specific Ift20 deletion with Col2-Cre or Aggrecan-CreERT2 drivers did not cause overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) resulted in shortened limb outgrowth. Although primary cilia were not formed in Ift20:Prx1-Cre mice, ciliary Hedgehog signaling was only moderately affected. Interestingly, loss of Ift20 lead to upregulation of Fgf18 expression resulting in ERK1/2 activation and sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate a novel mechanism of IFT20 in early chondrogenesis during endochondral ossification.

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The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport

eLife ◽

10.7554/elife.02419 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 45

Author(s):

Diego Huet ◽

Thierry Blisnick ◽

Sylvie Perrot ◽

Philippe Bastin

Keyword(s):

Trypanosoma Brucei ◽

Protein Complexes ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Small Gtpase ◽

Anterograde Transport ◽

Cargo Transport ◽

Cilia And Flagella ◽

Bidirectional Movement

The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.

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