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Blue Native PAGE co-fractionation of purified human endosomes

ABSTRACT: EndoMAP aims to systematically dissect the human endosomal protein complexes. To purify endosomes from human cells, a modified Endo-IP protocol was developed to be compatible with complexomics methodologies and evaluated by DIA-MS. Three independent biological replicates of human endosomes purified by the modified Endo-IP protocol were subjected to Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE) co-fractionation to identify possible protein complexes based on correlation profiling.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Miguel A. Gonzalez-Lozano

LAB HEAD: J. Wade Harper

PROVIDER: PXD054684 | Pride | 2025-03-29

REPOSITORIES: Pride

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Project description:Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and gene ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling.

			Action	DRS
	BN_DIA_Endo.pg_matrix.tsv	Tabular
	BN_DIA_Endo.tsv	Tabular
	BN_sample_metadata.xlsx	Xlsx
	EndoIP.pg_matrix.tsv	Tabular
	EndoIP.tsv	Tabular

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Blue Native PAGE co-fractionation of purified human endosomes

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