scholarly journals Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

2018 ◽  
Author(s):  
Shi-Rong Hong ◽  
Cuei-Ling Wang ◽  
Yao-Shen Huang ◽  
Yu-Chen Chang ◽  
Ya-Chu Chang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Living Cells ◽  
Comprehensive Understanding ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Dynamic Distribution ◽  
Wide Range

AbstractTubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here we develop a method to rapidly deplete tubulin glutamlyation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

scholarly journals IFT20 is critical for early chondrogenesis during endochondral ossification

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.

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

2019 ◽  
Author(s):  
Wei Wang ◽  
Bailey A. Allard ◽  
Tana S. Pottorf ◽  
Jay L. Vivian ◽  
Pamela V. Tran
Keyword(s):  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Double Mutant ◽  
Primary Cilia ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Ciliary Protein ◽  
Transport Defect

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.

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

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.

scholarly journals Intraflagellar transport is deeply integrated in hedgehog signaling

2018 ◽  
Vol 29 (10) ◽  
pp. 1178-1189 ◽  
Author(s):  
Thibaut Eguether ◽  
Fabrice P. Cordelieres ◽  
Gregory J. Pazour
Keyword(s):  
Transcription Factors ◽  
Membrane Protein ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Hedgehog Pathway ◽  
Gli Transcription Factors ◽  
Cilia Formation ◽  
Mutant Cells

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.

scholarly journals Uncoupling Intraflagellar Transport and Primary Cilia Formation Demonstrates Deep Integration of IFT in Hedgehog Signaling

2017 ◽  
Author(s):  
Thibaut Eguether ◽  
Fabrice P Cordelieres ◽  
Gregory J Pazour
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Embryonic Fibroblasts ◽  
Fk506 Binding Protein ◽  
Gli Transcription Factors ◽  
Cilia Formation ◽  
Deep Integration ◽  
Mutant Cells

AbstractThe 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 if 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 similar to other IFTs allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway inIft27mutant cells showed that IFT is extensively involved in the pathway. Similar analysis ofBbsmutant 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 stop IFT after ciliary assembly was complete we show that smoothened delivery to cilia is IFT independent.AbbreviationsMEFsmouse embryonic fibroblastsSAGsmoothen agonistIFTintraflagellar transportFKBPFK506 Binding Protein 12FRBFKBP12-rapamycin binding

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

2019 ◽  
Vol 9 (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.

scholarly journals DUBs Activating the Hedgehog Signaling Pathway: A Promising Therapeutic Target in Cancer

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1518
Author(s):  
Francesca Bufalieri ◽  
Ludovica Lospinoso Severini ◽  
Miriam Caimano ◽  
Paola Infante ◽  
Lucia Di Marcotullio
Keyword(s):  
Drug Targets ◽  
Hedgehog Signaling ◽  
Dynamic Balance ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Signaling Transduction ◽  
E3 Ligases ◽  
Stem Cell Maintenance ◽  
Wide Range ◽  
Potential Applications

The Hedgehog (HH) pathway governs cell proliferation and patterning during embryonic development and is involved in regeneration, homeostasis and stem cell maintenance in adult tissues. The activity of this signaling is finely modulated at multiple levels and its dysregulation contributes to the onset of several human cancers. Ubiquitylation is a coordinated post-translational modification that controls a wide range of cellular functions and signaling transduction pathways. It is mediated by a sequential enzymatic network, in which ubiquitin ligases (E3) and deubiquitylase (DUBs) proteins are the main actors. The dynamic balance of the activity of these enzymes dictates the abundance and the fate of cellular proteins, thus affecting both physiological and pathological processes. Several E3 ligases regulating the stability and activity of the key components of the HH pathway have been identified. Further, DUBs have emerged as novel players in HH signaling transduction, resulting as attractive and promising drug targets. Here, we review the HH-associated DUBs, discussing the consequences of deubiquitylation on the maintenance of the HH pathway activity and its implication in tumorigenesis. We also report the recent progress in the development of selective inhibitors for the DUBs here reviewed, with potential applications for the treatment of HH-related tumors.

Surface Structure of Nucleated Animal Cells: Carbon Replicas

1967 ◽  
Vol 25 ◽  
pp. 120-121
Author(s):  
Robert M. Glaeser ◽  
Thea B. Scott
Keyword(s):  
Cell Surface ◽  
Surface Structure ◽  
Particle Diameter ◽  
Cellular Functions ◽  
Animal Cells ◽  
Replica Technique ◽  
Carbon Replica ◽  
Characteristic Particle ◽  
Cell Surface Structure ◽  
Carbon Replicas

The carbon-replica technique can be used to obtain information about cell-surface structure that cannot ordinarily be obtained by thin-section techniques. Mammalian erythrocytes have been studied by the replica technique and they appear to be characterized by a pebbly or “plaqued“ surface texture. The characteristic “particle” diameter is about 200 Å to 400 Å. We have now extended our observations on cell-surface structure to chicken and frog erythrocytes, which possess a broad range of cellular functions, and to normal rat lymphocytes and mouse ascites tumor cells, which are capable of cell division. In these experiments fresh cells were washed in Eagle's Minimum Essential Medium Salt Solution (for suspension cultures) and one volume of a 10% cell suspension was added to one volume of 2% OsO4 or 5% gluteraldehyde in 0.067 M phosphate buffer, pH 7.3. Carbon replicas were obtained by a technique similar to that employed by Glaeser et al. Figure 1 shows an electron micrograph of a carbon replica made from a chicken erythrocyte, and Figure 2 shows an enlarged portion of the same cell.

Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

1992 ◽  
Vol 50 (1) ◽  
pp. 418-419
Author(s):  
D. L. Taylor
Keyword(s):  
Living Cells ◽  
Cellular Level ◽  
Molecular Techniques ◽  
Cellular Functions ◽  
Macromolecular Assemblies ◽  
Cell Functions ◽  
Molecular Events ◽  
Yield Information ◽  
Full Knowledge ◽  
Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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

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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