Dataset Information

Mass-spectrometry-based genetic analysis of lysosome-related organelle in the intestine of Caenorhabditis elegans

ABSTRACT: The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of important molecules; parts of its functions rely on a type of lysosome-related organelle known as gut granules. However, aspects of the intestine are not well understood, including the exporting of its molecules and that of the gut granules, in which only a few of the organelle’s protein content is identified. Here, we report a mass-spectrometry-based proteomic analysis of the intestine of the Caenorhabditis elegans and of these lysosome-related organelles. We identified around 5000 proteins each in the intestine and the gonad and showed that most of these proteins can be identified using samples extracted from a single worm, suggesting the feasibility of individual-level proteomic-based genetics analysis. Comparing the proteome to that of the transcriptome, we identify proteins that may be synthesized in the intestine and transferred to the gonad. To identify gut granule proteins, we compared the intestinal proteome of individual intestines that largely lack gut granules to that of the wild-type. The identified gut granule proteome includes proteins previously known to be exclusively localized to the granules, and we validated two more with immunohistochemistry analysis, including a worm ortholog of the mammalian ABCC6/MRP6, suggesting that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single tissue mass-spectrometry-based proteomic analyses in small organisms such as nematodes, as well as its utility for making discoveries in a previously prohibitive area.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Intestine, Gonad

SUBMITTER: Ting-Yu Wang

LAB HEAD: Tsui-Fen Chou

PROVIDER: PXD047792 | Pride | 2024-05-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	James_20220225-DDM-G1.xlsx	Xlsx
	James_20220225-DDM-G1_chimerys20230531.msf	Msf
	James_20220225_OTE_Aur_1hr_DDM-G1.raw	Raw
	James_20220225_OTE_Aur_1hr_DDM-G2.raw	Raw
	James_20220225_OTE_Aur_1hr_DDM-G3.raw	Raw

Items per page:

1 - 5 of 52

Similar Datasets

Project description:Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., {alpha}-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Total RNA was extracted from pooled small intestines of three WT and three CF mice using Tri-Reagent (Molecular Research Center, Cincinnati, OH), and poly(A) RNA was purified by using the MicroPoly(A) mRNA purification kit (Ambion, Austin, TX). The WT and CF poly(A) RNA samples were sent to IncyteGenomics (St. Louis, MO) where they were labeled with cyanine 3 (Cy3) and Cy5, respectively, and hybridized with the UniGEM1.31 array representing 9,570 known genes and expressed sequence tags.

Dataset Information

Mass-spectrometry-based genetic analysis of lysosome-related organelle in the intestine of Caenorhabditis elegans

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets