Dataset Information

Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

ABSTRACT: The associated files are the results of AP-MS experiments done from Xenopus multiciliated cells derived from dissected animal caps. Baits used for affinity purification were GFP-dnai2, GFP-wdr78, and GFP-C16orf71. Note that some files associated with the C16orf71 experiments may be mislabeled C16orf11 but reflect the C16orf71 protein nevertheless. Two sets of GFP pulldown controls were used for comparison, one for the dnai2 and wdr78 baits and a separate control for the C16orf71 experiment. The dnai2 and wdr78 sample sets were run on an Orbitrap Fusion and the C16orf71 samples were run on an Orbitrap Fusion Lumos mass spectrometer.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Xenopus Laevis (ncbitaxon:8355)

SUBMITTER: Edward Marcotte

PROVIDER: MSV000085075 | MassIVE | Tue Mar 10 12:25:00 GMT 2020

SECONDARY ACCESSION(S): PXD017980

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Publications

Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins.

Lee Chanjae C Cox Rachael M RM Papoulas Ophelia O Horani Amjad A Drew Kevin K Devitt Caitlin C CC Brody Steven L SL Marcotte Edward M EM Wallingford John B JB

eLife 20201202

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use <i>in vivo</i> imaging ...[more]

PMID: 33263282

Similar Datasets

Project description:The amyloid beta (Aβ) peptide represents a 37 to 49 amino acids endoproteolytic fragment of the amyloid precursor protein. The cellular biology that governs the formation and clearance of Aβ has been understood to play a critical role in Alzheimer’s disease (AD). The primary objective of this study was to generate an in-depth inventory of human brain proteins that oligomeric preparations of Aβ1-42 can bind to using an unbiased in vitro discovery approach. Synthetic Aβ1-42 peptides and a brain extract generated from adult human frontal lobe tissue served in these studies as baits and biological source material, respectively. oAβ1-42 was prepared by aggregating the peptide at 4 ºC for 24 h, using previously described procedures known to generate amyloid-β-derived diffusible ligands (ADDLs). Because the interaction with a given binding partner may rely on a binding epitope that comprises N- or C-terminal residues of Aβ1-42, two separate experiments (I and II) were conducted, which differed in the orientation the oAβ1-42 bait was tethered to the affinity matrix. To facilitate meaningful comparisons across experiments, the method of Aβ1-42 capture was not based on immunoaffinity reagents. Instead, alternative Aβ1-42 baits were equipped with biotin moieties attached to the N- or C-terminus by a 6-carbon linker chain, enabling their consistent affinity-capture on streptavidin agarose matrices. Biotin-saturated streptavidin agarose matrices served as negative controls and three biological replicates of samples and controls were generated for each of the three separate interactome datasets by reproducing the affinity-capture step side-by-side on three separate streptavidin agarose affinity matrices that had been saturated with the biotinylated baits. To identify differences in protein-protein interactions of monomeric versus oligomeric Aβ1-42, a 3rd interactome experiment was conducted in which oAβ1-42-biotin or mAβ1-42-biotin served as baits. Digitonin-solubilized brain extracts, which are known to comprise extracellular and most cellular proteins (except for nuclear proteins) served as biological starting material, consistent with the main subcellular areas previously reported to harbor Aβ. Following extensive washes of affinity matrices in their protein-bound state, binders to the bait peptides were eluted by rapid acidification, fully denatured in 9 M urea, and trypsinized. To avoid notorious confounders related to variances in the subsequent handling and analysis of samples, individual peptide mixtures were labeled with distinct isobaric tandem mass tags (TMT) in a six-plex format, then combined and concomitantly subjected to ZipTip-based pre-analysis clean-up by strong cation exchange (SCX) and reversed phase (RP) separation. Four-hour split-free reversed phase nanospray separations were online coupled to an Orbitrap Fusion Tribrid mass spectrometer, which was configured to run an MS3 analysis method.