The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.

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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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A rapid and affordable screening platform for membrane protein trafficking

BMC Biology ◽

10.1186/s12915-015-0216-3 ◽

2015 ◽

Vol 13 (1) ◽

Cited By ~ 17

Author(s):

Joshua C. Snyder ◽

Thomas F. Pack ◽

Lauren K. Rochelle ◽

Subhasish K. Chakraborty ◽

Ming Zhang ◽

...

Keyword(s):

Membrane Protein ◽

Protein Trafficking ◽

Membrane Protein Trafficking ◽

Screening Platform

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Ethanol and Membrane Protein Trafficking: Diverse Mechanisms of Ethanol Action

Alcoholism Clinical and Experimental Research ◽

10.1111/j.1530-0277.2002.tb02536.x ◽

2002 ◽

Vol 26 (2) ◽

pp. 287-293 ◽

Cited By ~ 10

Author(s):

Laura E. Nagy ◽

M. Raj Lakshman ◽

Carol A. Casey ◽

Cynthia F. Bearer

Keyword(s):

Membrane Protein ◽

Protein Trafficking ◽

Membrane Protein Trafficking

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Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e12-01-0047 ◽

2012 ◽

Vol 23 (15) ◽

pp. 2917-2929 ◽

Cited By ~ 55

Author(s):

Emily Deutsch ◽

Aubrey V. Weigel ◽

Elizabeth J. Akin ◽

Phil Fox ◽

Gentry Hansen ◽

...

Keyword(s):

Membrane Protein ◽

Cell Surface ◽

Ion Channel ◽

Protein Trafficking ◽

Hippocampal Neurons ◽

Surface Membrane ◽

Cell Types ◽

Homogeneous Surface ◽

Hek Cells ◽

Membrane Protein Trafficking

Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.

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Non-cell autonomous inhibition of the Shh pathway due to impaired cholesterol biosynthesis requires Ptch1/2

10.1101/2020.12.10.420588 ◽

2020 ◽

Author(s):

Carian Jägers ◽

Henk Roelink

Keyword(s):

Sonic Hedgehog ◽

Birth Defects ◽

Cholesterol Synthesis ◽

Cholesterol Biosynthesis ◽

Shh Signaling ◽

Shh Pathway ◽

Opitz Syndrome ◽

Cholesterol Precursors ◽

G Protein Coupled ◽

Mutant Cells

AbstractBirth defects due to congenital errors in enzymes involved cholesterol synthesis like Smith-Lemli-Opitz syndrome (SLOS) and Lathosterolosis cause an accumulation of cholesterol precursors and a deficit in cholesterol. The phenotype of both SLOS and Lathosterolosis have similarities to syndromes associated with abnormal Sonic hedgehog (Shh) signaling, consistent with the notion that impaired cholesterol signaling can cause reduced Shh signaling. Two multipass membrane proteins play central roles in Shh signal transduction, the putative Resistance, Nodulation and Division (RND) antiporters Ptch1 and Ptch2, and the G-protein coupled receptor Smoothened (Smo). Sterols have been suggested as cargo for Ptch1, while Smo activity can affected both positively and negatively by steroidal molecules. We demonstrate that mESCs mutant for 7-dehydroxycholesterol reductase (7dhcr) or sterol-C5-desaturase (sc5d) reduce the Hh response in nearby wildtype cells when grown in mosaic organoids. This non-cell autonomous inhibitory activity of the mutant cells required the presence of both Ptch1 and Ptch2. These observations support a model in which late cholesterol precursors that accumulate in cells lacking 7DHCR are the cargo for Ptch1 and Ptch2 activity that mediates the non-cell autonomous inhibition of Smo.

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Fluorogenic Probing of Membrane Protein Trafficking

Bioconjugate Chemistry ◽

10.1021/acs.bioconjchem.8b00180 ◽

2018 ◽

Vol 29 (6) ◽

pp. 1823-1828 ◽

Cited By ~ 11

Author(s):

Chenge Li ◽

Aurélien Mourton ◽

Marie-Aude Plamont ◽

Vanessa Rodrigues ◽

Isabelle Aujard ◽

...

Keyword(s):

Membrane Protein ◽

Protein Trafficking ◽

Membrane Protein Trafficking

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Insights in how amphetamine ROCKs (Rho-associated containing kinase) membrane protein trafficking

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520960112 ◽

2015 ◽

Vol 112 (51) ◽

pp. 15538-15539

Author(s):

Christine Saunders ◽

Aurelio Galli

Keyword(s):

Membrane Protein ◽

Protein Trafficking ◽

Membrane Protein Trafficking

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Gli2, but notGli1, is required for initial Shh signaling and ectopic activation of the Shh pathway

Development ◽

10.1242/dev.129.20.4753 ◽

2002 ◽

Vol 129 (20) ◽

pp. 4753-4761 ◽

Cited By ~ 35

Author(s):

C. Brian Bai ◽

Wojtek Auerbach ◽

Joon S. Lee ◽

Daniel Stephen ◽

Alexandra L. Joyner

Keyword(s):

Signaling Pathway ◽

Negative Regulator ◽

Embryonic Patterning ◽

Mouse Development ◽

Shh Signaling ◽

Shh Pathway ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Mammalian Tissues ◽

Ectopic Activation

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, we generated a Gli1 null allele expressing lacZ. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, we show that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. We show that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Of significance, we found instead that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. Our studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc.

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