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Analysis of the cilia membrane proteome by cmAPEX

ABSTRACT: APEX was targeted to the primary cilium of mouse IMCD cells using a 5HT6-APEX-GFP fusion construct. Proximity labling was performed using incubation with biotinphenol and H2O2. For details, please see the methods section of the accompanying paper.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Kidney

DISEASE(S): Mixed Disorder As Reaction To Stress

SUBMITTER: Markus Rinschen

LAB HEAD: Bernhard Schermer

PROVIDER: PXD005201 | Pride | 2017-07-18

REPOSITORIES: Pride

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The ciliary membrane-associated proteome reveals actin-binding proteins as key components of cilia.

Kohli Priyanka P Höhne Martin M Jüngst Christian C Bertsch Sabine S Ebert Lena K LK Schauss Astrid C AC Benzing Thomas T Rinschen Markus M MM Schermer Bernhard B

EMBO reports 20170714 9

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification ...[more]

PMID: 28710093

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MicroRNA (miRNA) expression of IMCD tubule suspension

Project description:miRNA plays a role as post-transcriptional regulator. However, miRNAs in the kidney collecting duct cell have not been well understood. So we aimed to profile miRNAs in the kidney inner medullary collecting duct (IMCD) cells, and to identify the vasopressin-responsive miRNAs in the kidney IMCD cells. The microarray assay revealed that relative expression of miRNAs in the kidney IMCD cells was changed by desmopressin (dDAVP) stimulation. Fresh IMCD tubules were prepared from rat kidney and incubated in the medium in the absence or presence of dDAVP (1 nM, 2 h). Total RNA purified from IMCD tubules was used to analyze miRNA expression by GeneChipM-BM-. miRNA 3.0 Array (Affymetrix).

2014-06-21 | E-GEOD-58693 | biostudies-arrayexpress

Primary cilia control translation and the cell cycle in medulloblastoma

Project description:The primary cilium, a signaling organelle projecting from the surface of a cell, controls cellular physiology and behavior. The presence or absence of primary cilia is a distinctive feature of a given tumor type; however, whether and how the primary cilium contributes to tumorigenesis is unknown for most tumors. Medulloblastoma (MB) is a common pediatric brain cancer comprising four groups: SHH, WNT, group 3 (G3), and group 4 (G4). From 111 cases of MB, we show that primary cilia are abundant in SHH and WNT MBs but rare in G3 and G4 MBs. Using WNT and G3 MB mouse models, we show that primary cilia promote WNT MB by facilitating translation of mRNA encoding β-catenin, a major oncoprotein driving WNT MB, whereas cilium loss promotes G3 MB by disrupting cell cycle control and destabilizing the genome. Our findings reveal tumor type–specific ciliary functions and underlying molecular mechanisms. Moreover, we expand the function of primary cilia to translation control and reveal a molecular mechanism by which cilia regulate cell cycle progression, providing new frameworks for studying cilia function in normal and pathologic conditions.

2022-07-04 | GSE207008 | GEO

Loss of primary cilia drives switching from Hedgehog to Ras/MAPK pathway

Project description:Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naïve tumors responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains frequent. While the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, with the molecular basis of tumor type and pathway selection still obscure. Here we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoi-resistant BCCs possess an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared to naive or Gorlin patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature reveals increased Ras/MAPK pathway activation. Tissue analysis confirms an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiates Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines confers resistance to both canonical (vismodegib) and non-canonical (aPKC and MRTF inhibitors) HH pathway inhibitors, while conferring sensitivity to MAPK inhibitors. Our results provide new insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH to Ras/MAPK pathway switching.

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Longitudinal study of the small urinary extracellular vesicle proteome during living donor kidney transplantation

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2021-08-30 | PXD005219 | Pride

Toxoplasma GRA protein identification

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2018-11-02 | PXD009604 | Pride

Piwi is a key regulator of both somatic and germline stem cells in the Drosophila testis

Project description:The Piwi-piRNA pathway is well known for its germline function, yet its somatic role remains elusive. We show here that Piwi is required autonomously not only for germline stem cell (GSC) but also for somatic cyst stem cell (CySC) maintenance in the Drosophila testis. Reducing Piwi activity in the testis caused defects in CySC differentiation. Accompanying this, GSC daughters expanded beyond the vicinity of the hub but failed to differentiate further. Moreover, Piwi deficient in nuclear localization caused similar defects in somatic and germ cell differentiation, which was rescued by somatic Piwi expression. To explore the underlying molecular mechanism, we identified Piwi-bound piRNAs that uniquely map to a gene key for gonadal development, Fasciclin 3, and demonstrate that Piwi regulates its expression in somatic cyst cells. Our work reveals the cell-autonomous function of Piwi in both somatic and germline stem cell types, with somatic function possibly via its epigenetic mechanism. Examination of Piwi-piRNAs in fly testis

2015-05-23 | E-GEOD-68997 | biostudies-arrayexpress

Transcription profiling of rat inner medullary collecting duct

Project description:Identification of gene expressed in the enriched inner medullary collecting duct cells in rat. Experiment Overall Design: Rat inner medullary collecting duct (IMCD) cells were isolated from 7 male Sprage-Dawley rats by collagenase and hyaluronidase digestion and follow by low speed centrifugation. The non-IMCD cells were collected by centrifugation of supernatant of enriched IMCD samples. Experiment Overall Design: Total RNA about 3 ug were used per microarray (Rat 230 2.0 Genechip array). Experiment Overall Design: The experiments were repeat 3 times (3 pairs of IMCD VS non-IMCD)

2008-06-15 | E-GEOD-7891 | biostudies-arrayexpress

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Project description:APEX proteomics data of the vesicular proteome of the T cell inhibitory receptors CTLA-4, LAG3 and TIM3 as described in https://biorxiv.org/cgi/content/short/2023.07.21.550019v1

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The lymph node transcriptome of unicentric and idiopathic multicentric Castleman disease

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Osmolality as a major regulator of the transcriptome and metabolome in kidney collecting duct cells

Project description:We hypothesized that altered extracellular osmolality per se could affect the transcriptome of the kidney inner medullary collecting duct (IMCD) cells, and hence it might change renal tubular function. The data sets of transcriptomics were incorporated into the "omic" data sets of metabolomics. Primary cultured IMCD cells of rat kidney were grown in hyperosmolar culture medium (640 mOsm/KgH2O) for 4 d, and then the cells were cultured in the medium with either reduced (300 mOsm/KgH2O) or the same osmolality for 1 or 2 d more. Osmolality stress response. Total RNA from IMCD cells exposed to 300 or 640 mOsm/KgH2O for 24 h (n=3 in each group, 24 h-300 and 24 h-640) or for 48 h (n=3 in each group, 48 h-300 and 48 h-640) after a 4-day-culture in culture media (640 mOsm/KgH2O) were subjected to transcriptome analysis (Affymetrix GeneChip® Rat Gene 1.0 ST array containing 29,215 gene-level probe sets) to profile the gene expression at the mRNA level.

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