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Mass spectrometry analysis of kinetochore proteins (KKT4, KKT14, and KKT22) in Trypanosoma brucei

ABSTRACT: We performed immunoprecipitation and mass spectrometry of kinetoplastid kinetochore proteins (full-length proteins for KKT4, KKT14, and KKT22 as well as KKT14 fragments) to identify co-purifying proteins.

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ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Bungo Akiyoshi

LAB HEAD: Bungo Akiyoshi

PROVIDER: PXD047806 | Pride | 2024-01-18

REPOSITORIES: Pride

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Project description:The vertebrate retina uses diverse neuronal cell types arrayed into complex neural circuits to extract, process and relay information from the visual scene to the higher order processing centers of the brain. Amacrine cells, a diverse class of inhibitory interneurons, are thought to mediate the majority of the processing of the visual signal that occurs within the retina. Despite morphological characterization, the number of known molecular markers of amacrine cell types is still much smaller than the 26 morphological types that have been identified. Furthermore, it is not known how this diversity arises during development. Here, we have combined in vivo genetic labeling and single cell genome-wide expression profiling to: 1) Identify specific molecular types of amacrine cells; 2) Demonstrate the molecular diversity of the amacrine cell class. It is difficult to identify new markers of amacrine cells, due to the fact that they only comprise a small percentage of the total cells in the retina. Additionally, given that there are at least 26 distinct types of amacrine cells, population based approaches fail to achieve the precision necessary to discover markers of each type. To facilitate the identification of new markers for different amacrine cell classes and to more fully characterize the molecular signatures of these classes, we isolated single amacrine cells. To accomplish this goal, we introduced genetic reporters (pNdrg4::GFP or pSynapsin::GFP) into the developing retina (P0) by either in vivo or ex vivo electroporation. These reporters were observed to label morphologically distinct sets of amacrine cells at the different timepoints harvested in this study. Electroporated retinas were then dissociated at different time points and single retinal amacrine cells were isolated by virtue of their GFP expression and placed in tubes containing lysis buffer. Then, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software.

			Action	DRS
	GFP-KKT14C_BA.raw	Raw
	GFP-KKT14N_1NM-PP1_rep1.raw	Raw
	GFP-KKT14N_1NM-PP1_rep2.raw	Raw
	GFP-KKT14N_1NM-PP1_rep3.raw	Raw
	GFP-KKT14N_BA.raw	Raw

Dataset Information

Mass spectrometry analysis of kinetochore proteins (KKT4, KKT14, and KKT22) in Trypanosoma brucei

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