Proteomics of Primary Cilia by Proximity Labeling

Mick, David U.; Rodrigues, Rachel B.; Leib, Ryan D.; Adams, Christopher M.; Chien, Allis; Gygi, Steven P.; Nachury, Maxence V.

doi:10.1016/j.devcel.2015.10.015

Cited by 355 publications

(435 citation statements)

References 78 publications

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“…Until recently, PKA was thought to phosphorylate Gli proteins at the base of cilia under basal or SHH-off conditions due to PKA-C localization there (5). Several PKA subunits were recently detected within the cilium using cilia proximity labeling, and PKA-R1α was detected within cilia using immunofluorescence (6). Additionally, by specifically inhibiting PKA activity within cilia, Mick et al found that ciliary PKA activity is necessary for proper Gli3 protein processing (6).…”

Section: Resultsmentioning

confidence: 99%

“…Several PKA subunits were recently detected within the cilium using cilia proximity labeling, and PKA-R1α was detected within cilia using immunofluorescence (6). Additionally, by specifically inhibiting PKA activity within cilia, Mick et al found that ciliary PKA activity is necessary for proper Gli3 protein processing (6). We targeted a PKA activity sensor, AKAR4 (19), to the cilia using the 5HT6 receptor (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Mick et al have shown that SAG-mediated reduction of the Gli3 repressor is dependent on ciliary PKA activity (6). Although it has been speculated that stimulation of the HH pathway leads to decreased PKA activity, this link has not been tested within cilia.…”

Section: Shh Increases Camentioning

confidence: 99%

“…PKA activity is thus critical for HH signaling, particularly in maintaining the basal SHH-off state. PKA has been thought to regulate Gli proteins at the base of the cilium (5); however, recently, the subunits of PKA have been detected in cilia (6).…”

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confidence: 99%

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Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Moore

Stepanchick

Tewson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

108

133

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Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Shh Increases Camentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Moore

Stepanchick

Tewson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

108

133

View full text Add to dashboard Cite

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“…MiniSOG is a fluorescent flavoprotein that, when genetically encoded, is used as a versatile tag that emits fluorescence and, in response to illumination, generates singlet oxygen that transforms diaminobenzidine into electron-dense precipitates that can by imaged by using EM. The engineered peroxidase APEX, by contrast, can be used for EM and proximity labeling upon its fusion to a protein of interest, (Mick et al, 2015). Alternatively, events or regions of interest can be tracked over the two imaging modalities, with recording of coordinates imprinted on the culture dish, by using either commercialized (Al Jord et al, 2014) or lasermicropatterned culture substrates (Spiegelhalter et al, 2010).…”

Section: Ground-breaking In Vitro Clemmentioning

confidence: 99%

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Follain

Mercier

Osmani

et al. 2016

Journal of Cell Science

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Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, superresolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

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Proteomic Identification Reveals the Role of Ciliary Extracellular‐Like Vesicle in Cardiovascular Function

et al. 2020

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Primary cilia are shown to have membrane swelling, also known as ciliary bulbs. However, the role of these structures and their physiological relevance remains unknown. Here, it is reported that a ciliary bulb has extracellular vesicle (EV)‐like characteristics. The ciliary extracellular‐like vesicle (cELV) has a unique dynamic movement and can be released by mechanical fluid force. To better identify the cELV, differential multidimensional proteomic analyses are performed on the cELV. A database of 172 cELV proteins is generated, and all that examined are confirmed to be in the cELV. Repressing the expression of these proteins in vitro and in vivo inhibits cELV formation. In addition to the randomized heart looping, hydrocephalus, and cystic kidney in fish, compensated heart contractility is observed in both fish and mouse models. Specifically, low circulation of cELV results in hypotension with compensated heart function, left ventricular hypertrophy, cardiac fibrosis, and arrhythmogenic characteristics, which result in a high mortality rate in mice. Furthermore, the overall ejection fraction, stroke volume, and cardiac output are significantly decreased in mice lacking cELV. It is thus proposed that the cELV as a nanocompartment within a primary cilium plays an important role in cardiovascular functions.

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Proteomics of Primary Cilia by Proximity Labeling

Cited by 355 publications

References 78 publications

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Proteomic Identification Reveals the Role of Ciliary Extracellular‐Like Vesicle in Cardiovascular Function

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