scholarly journals Ciliary IFT88 safeguards coordinated epiphyseal vascularisation, resorption and ossification from disruptive physiological mechanical forces

2021 ◽  
Author(s):  
Clarissa R Coveney ◽  
Jasmine Samvelyan ◽  
Jadwiga Miotla-Zarebska ◽  
Josephine Carnegie ◽  
Emer Chang ◽  
...  
Keyword(s):  
Growth Plate ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Skeletal Maturation ◽  
Mechanical Forces ◽  
Dependent Manner ◽  
Pivotal Phase ◽  
Hypertrophic Chondrocyte ◽  
Physiological Loading ◽  
Tissue Responses

In the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. Despite this, how mechanical cues are integrated into biological programmes remains unclear. Primary cilia are microtubule-based organelles that tune a range of cell activities, including signalling cascades activated or modulated, by extracellular biophysical cues. Here, we demonstrate that the inducible, cartilage-specific deletion of Intraflagellar transport protein 88 (IFT88), which reduces ciliation in the adolescent mouse growth plate (GP), uncouples chondrocyte differentiation from cartilage resorption and mineralisation in a mechano-dependent manner. Targeting IFT88, inhibits hypertrophic chondrocyte VEGF expression, vascular recruitment, osteoclastic activity and the replacement of cartilage with bone. These effects are largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. Increases in physiological loading, in control mice, also impairs ossification in the peripheral GP, mimicking the effects of IFT88 deletion. Strikingly, limb immobilisation rescues disrupted VEGF and restores epiphyseal dynamics in Ift88cKO mice. These data indicate, that during this pivotal phase in adolescent skeletal maturation that defines the cessation of growth, ciliary IFT88 protects the coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces.

scholarly journals Microtubule stabilization drives 3D centrosome migration to initiate primary ciliogenesis

2017 ◽  
Vol 216 (11) ◽  
pp. 3713-3728 ◽  
Author(s):  
Amandine Pitaval ◽  
Fabrice Senger ◽  
Gaëlle Letort ◽  
Xavier Gidrol ◽  
Laurent Guyon ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Mechanical Forces ◽  
Apical Surface ◽  
Microtubule Network ◽  
Microtubule Stabilization ◽  
Cytoskeletal Remodeling ◽  
Cell Architecture ◽  
Cytoskeleton Remodeling ◽  
Insight Into

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface, yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetrical contraction participate in concert to drive apical centrosome migration. The distal appendage protein Cep164 appears to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas intraflagellar transport 88’s role seems to be restricted to axoneme elongation. Together, our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.

scholarly journals Intraflagellar transport-A deficiency attenuates ADPKD in a renal tubular- and maturation-dependent manner

2020 ◽  
Author(s):  
Wei Wang ◽  
Luciane M. Silva ◽  
Henry H. Wang ◽  
Matthew A. Kavanaugh ◽  
Tana S. Pottorf ◽  
...  
Keyword(s):  
Mouse Models ◽  
Primary Cilia ◽  
Protein Complexes ◽  
Collecting Duct ◽  
Intraflagellar Transport ◽  
Renal Tubules ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Knock Out

AbstractPrimary cilia are sensory organelles that are built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of certain ciliary genes in Autosomal Dominant Polycystic Kidney Disease (ADPKD) mouse models markedly attenuates PKD severity, indicating that a component of cilia dysfunction may have potential therapeutic value. To broaden the role of ciliary dysfunction, here we investigate the role of global deletion of Ift-A gene, Thm1, in juvenile and adult ADPKD mouse models. In cyst-lining cells of both juvenile and adult ADPKD models, cortical collecting duct cilia lengths and cytoplasmic and nuclear levels of the nutrient sensor, O-linked β-Nacetylglucosamine (O-GlcNAc) were increased. Relative to juvenile Pkd2 conditional knock-out mice, deletion of Thm1 together with Pkd2 both increased and reduced cystogenesis in a tubule-specific manner without altering kidney function, inflammation, cilia lengths, and ERK, STAT3 and OGlcNAc signaling. In contrast, Thm1 deletion in adult ADPKD mouse models markedly attenuated almost all features of PKD, including renal cystogenesis, inflammation, cilia lengths, and ERK, STAT3 and O-GlcNAc signaling. These data suggest that differential factors in the microenvironments between renal tubules and between developing and mature kidneys influence cilia and ADPKD pathobiology. Further, since O-GlcNAcylation directly regulates ciliary homeostasis and the balance between glycolysis and oxidative phosphorylation, we propose that increased O-GlcNAcylation may promote certain key ADPKD pathological processes.

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.

scholarly journals Nucleus pulposus primary cilia alter their length in response to changes in extracellular osmolarity but do not control TonEBP-mediated osmoregulation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hyowon Choi ◽  
Vedavathi Madhu ◽  
Irving M. Shapiro ◽  
Makarand V. Risbud
Keyword(s):  
Nucleus Pulposus ◽  
Primary Cilia ◽  
Target Genes ◽  
Intraflagellar Transport ◽  
Proximal Promoter ◽  
Null Mouse ◽  
Primary Role ◽  
Transactivation Domain ◽  
Enhancer Binding Protein ◽  
Wild Type Mefs

Abstract The nucleus pulposus (NP) cells adapt to their physiologically hyperosmotic microenvironment through Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cell5 [NFAT5])-mediated osmoregulation. Primary cilia in different organs serve diverse roles including osmosensing, but its contribution to NP cell osmoadaptive response is unknown. A high percentage of cultured primary NP cells possessed primary cilia that changed length in response to osmotic stimuli. Stable silencing of Intraflagellar Transport 88 (Ift88) or Kinesin Family Member 3 A (Kif3a) to inhibit the formation of primary cilia did not affect hyperosmotic upregulation of TonEBP. While ShKif3a blocked hyperosmotic increase of TonEBP-Transactivation Domain (TAD) activity, overall the knockdown of either gene did not alter the hyperosmotic status of proximal promoter activities and transcription of key TonEBP targets. On the other hand, a small decrease in TonEBP level under hypoosmotic condition was attenuated by Ift88 or Kif3a knockdown. Noteworthy, none of the TonEBP target genes were responsive to hypoosmotic stimulus in control and Ift88 or Kif3a knockdown cells, suggesting the primary role of TonEBP in the hyperosmotic adaptation of NP cells. Similarly, in Kif3a null mouse embryonic fibroblasts (MEFs), the overall TonEBP-dependent hyperosmotic responses were preserved. Unlike NP cells, TonEBP targets were responsive to hypoosmolarity in wild-type MEFs, and these responses remained intact in Kif3a null MEFs. Together, these results suggest that primary cilia are dispensable for TonEBP-dependent osmoadaptive response.

scholarly journals Long Term Cyclic Pamidronate Reduces Bone Growth by Inhibiting Osteoclast Mediated Cartilage-to-Bone Turnover in the Mouse

2008 ◽  
Vol 2 (1) ◽  
pp. 121-125 ◽  
Author(s):  
K.D Evans ◽  
L.E Sheppard ◽  
D.I Grossman ◽  
S.H Rao ◽  
R.B Martin ◽  
...  
Keyword(s):  
Bone Turnover ◽  
Growth Plate ◽  
Bone Growth ◽  
Cathepsin K ◽  
Chondrocyte Apoptosis ◽  
Cell Numbers ◽  
Hypertrophic Chondrocyte ◽  
Osteoclast Function ◽  
And Function

Bisphosphonates, used to treat diseases exhibiting increased osteoclast activity, reduce longitudinal bone growth through an as yet undefined mechanism. Pamidronate, an aminobisphosphonate, was given weekly to mice at 0, 1.25, or 2.50 mg/kg/wk beginning at 4 weeks of age. At 12 weeks of age, humeral length, growth plate area, regional chondrocyte cell numbers, chondrocyte apoptosis, TRAP stained osteoclast number, and osteoclast function assessed by cathepsin K immunohistochemistry were quantified. Humeral length was decreased in pamidronate treated mice compared to vehicle control mice, and correlated with greater growth plate areas reflecting greater proliferative and hypertrophic chondrocyte cell numbers with fewer hypertrophic cells undergoing apoptosis. Pamidronate treatment increased TRAP stained osteoclast numbers yet decreased cathepsin K indicating that pamidronate repressed osteoclast maturation and function. The data suggest that long term cyclic pamidronate treatment impairs bone growth by inhibition of osteoclast maturation thereby reducing cartilage-to-bone turnover within the growth plate.

scholarly journals Primary cilia of human endothelial cells disassemble under laminar shear stress

2004 ◽  
Vol 164 (6) ◽  
pp. 811-817 ◽  
Author(s):  
Carlo Iomini ◽  
Karla Tejada ◽  
Wenjun Mo ◽  
Heikki Vaananen ◽  
Gianni Piperno
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Primary Cilia ◽  
Umbilical Vein ◽  
Intraflagellar Transport ◽  
Mechanical Stimuli ◽  
Laminar Shear Stress ◽  
Human Endothelial Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Laminar Shear

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

scholarly journals Expression of IFT140 During Bone Development

2019 ◽  
Vol 67 (10) ◽  
pp. 723-734
Author(s):  
Chenyang Zhang ◽  
Shuai Zhang ◽  
Yao Sun
Keyword(s):  
Primary Cilia ◽  
Soft Tissues ◽  
Core Protein ◽  
Bone Development ◽  
Mrna Level ◽  
Intraflagellar Transport ◽  
Expression Level ◽  
Cell Functions ◽  
Extracellular Stimuli ◽  
Molecular Signals

Primary cilia, hair-like organelles projecting from the surface of cells, are critical for sensing extracellular stimuli and transmitting molecular signals that regulate cell functions. During bone development, cell cilia are found in several types of cells, but their roles require further investigation. Intraflagellar transport (IFT) is essential for the formation and maintenance of most eukaryotic cilia. IFT140 is a core protein of the IFT-A complex. Mutations in IFT140 have been associated with cases of skeletal ciliopathies. In this study, we examined the expression of IFT140 during bone development. The results showed that, compared with many soft tissues, Ift140 (mRNA level) was highly expressed in bone. Moreover, its expression level was downregulated in the long bones of murine osteoporosis models. At the histological level, IFT140 was characteristically expressed in osteoblasts and chondrocytes at representative stages of bone development, and its expression level in these two types of cells was observed in two waves. These findings suggest that IFT140 may play an important role in the process of chondrogenic and osteogenic differentiation during bone development.

scholarly journals Integrin-β1 is required for the renal cystogenesis caused by ciliary defects

2020 ◽  
Vol 318 (5) ◽  
pp. F1306-F1312
Author(s):  
Miran Yoo ◽  
Laura M. C. Barisoni ◽  
Kyung Lee ◽  
G. Luca Gusella
Keyword(s):  
Inflammatory Infiltrate ◽  
Primary Cilia ◽  
Genetic Model ◽  
Renal Cysts ◽  
Intraflagellar Transport ◽  
The Other ◽  
Integrin Β1 ◽  
Principal Cells ◽  
Other Hand ◽  
Collecting Ducts

Defects in the function of primary cilia are commonly associated with the development of renal cysts. On the other hand, the intact cilium appears to contribute a cystogenic signal whose effectors remain unclear. As integrin-β1 is required for the cystogenesis caused by the deletion of the polycystin 1 gene, we asked whether it would be similarly important in the cystogenetic process caused by other ciliary defects. We addressed this question by investigating the effect of integrin-β1 deletion in a ciliopathy genetic model in which the Ift88 gene, a component of complex B of intraflagellar transport that is required for the proper assembly of cilia, is specifically ablated in principal cells of the collecting ducts. We showed that the renal cystogenesis caused by loss of Ift88 is prevented when integrin-β1 is simultaneously depleted. In parallel, pathogenetic manifestations of the disease, such as increased inflammatory infiltrate and fibrosis, were also significantly reduced. Overall, our data indicate that integrin-β1 is also required for the renal cystogenesis caused by ciliary defects and point to integrin-β1-controlled pathways as common drivers of the disease and as possible targets to interfere with the cystogenesis caused by ciliary defects.

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

2019 ◽  
Vol 30 (7) ◽  
pp. 828-837 ◽  
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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