Dataset Information

MS analysis of purified TRIM46, 480AnkG and 480AnkG-NN.

ABSTRACT: Mass spectrometry analysis of purified GFP-TRIM46, 480AnkG-GFP and 480AnkG-NN-GFP.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Riccardo Stucchi

LAB HEAD: Casper Hoogenraad

PROVIDER: PXD013685 | Pride | 2019-08-30

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180716_OR12_Ag4_Stucc001_SA_480AnkG-GFP.msf	Msf
	20180716_OR12_Ag4_Stucc001_SA_480AnkG-GFP.raw	Raw
	20180716_OR12_Ag4_Stucc001_SA_480AnkG-GFP.xlsx	Xlsx
	20180716_OR12_Ag4_Stucc001_SA_480AnkG-NN-GFP.msf	Msf
	20180716_OR12_Ag4_Stucc001_SA_480AnkG-NN-GFP.raw	Raw

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Publications

Feedback-Driven Assembly of the Axon Initial Segment.

Fréal Amélie A Rai Dipti D Tas Roderick P RP Pan Xingxiu X Katrukha Eugene A EA van de Willige Dieudonnée D Stucchi Riccardo R Aher Amol A Yang Chao C Altelaar A F Maarten AFM Vocking Karin K Post Jan Andries JA Harterink Martin M Kapitein Lukas C LC Akhmanova Anna A Hoogenraad Casper C CC

Neuron 20190829 2

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46 ...[more]

PMID: 31474508

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Project description:During murine hypothalamic development, different neuroendocrine cell phenotypes are generated in overlapping periods; this suggests that cell-type specific developmental programs operate to achieve complete maturation. A balance between programs that include cell proliferation, cell cycle withdrawal as well as epigenetic regulation of gene expression characterizes neurogenesis. Thyrotropin releasing hormone (TRH) is a peptide that regulates energy homeostasis and autonomic responses. To better understand the molecular mechanisms underlying TRH neuron development, we performed a genome wide study of its transcriptome during fetal hypothalamic development. In primary cultures, TRH cells constitute 2% of the total fetal hypothalamic cell population. To purify these cells, we took advantage of the fact that the segment spanning –774 to +84 bp of the Trh gene regulatory region confers specific expression of the green fluorescent protein (GFP) in the TRH cells. Transfected TRH cells were purified by fluorescence activated cell sorting, various cell preparations pooled, and their transcriptome compared to that of GFP- hypothalamic cells. TRH cells undergoing the terminal phase of differentiation, expressed genes implicated in protein biosynthesis, intracellular signaling and transcriptional control. Among the transcription-associated transcripts, we identified the transcription factors Klf4, Klf10 and Atf3, which were previously uncharacterized within the hypothalamus. To our knowledge, this is the first report identifying transcripts with a potentially important role during the development of a specific hypothalamic neuronal phenotype. This genome-scale study forms a rational foundation for identifying genes that might participate in the development and function of hypothalamic TRH neurons. Hypothalamic primary cultures were prepared from E17 rat embryos. Twenty-four hours after seeding, cells were transfected using the minimal Trh promoter (-776/+84 bp) upstream of the GFP. Forty-eight hours after transfection, cells were trypsinized and subjected to FACS. TRH+ cells were purified from a pool of five 60-mm dishes using the FACS Vantage (Becton Dickinson, San Jose, CA) and the exclusion method at high speed (60 µl/min). In general, 20,000 GFP+ cells were purified from 5 X 106 cells. The microarray analysis was performed as described in the Affymetrix expression analysis technical manual (http://www.affymetrix.com). Total RNA (10 µg) was extracted from three different cell populations: i) sorted TRH-GFP+ cells (GFP+); ii) TRH-GFP+ and GFP- mixed cells (GFP+/-) passed through the FACS but not sorted, and iii) non transfected cells (NT). To obtain a sufficient amount of RNA for each cell population, the number of independent experiments pooled for the GFP+ sample was higher than for the other samples. Therefore, a pool of six independent experiments was used to prepare total RNA from the GFP+ and three independent experiments for GFP+/- or NT cells.