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The proteome of Tunneling Nanotubes (TNTs) in U2OS cells; comparison with Extracellular Vesicles (EVs)

ABSTRACT: Tunneling nanotubes (TNTs) are thin F-actin based membranous structures that form continuous cytoplasmic bridges between cells over distances ranging from several hundred nm up to 100 µm. They allow cell-to-cell communication by facilitating the transfer of different cargoes directly from cytoplasma to cytoplasma of the connected cells, including organelles (lysosomes, mitochondria), micro or mRNAs, pathogens, and misfolded proteins (for example prion proteins, tau or α-synuclein aggregates). TNTs could play major roles in various diseases, including neurodegenerative diseases or cancers of different types. TNT formation is highly dynamic and depends on cellular stresses and actin regulators. However, the mechanisms of TNT formation and TNT molecular components and regulators are still poorly understood. Because of their size, of some common components, of the ability to transfer cellular material to remote cells and because TNTs are fragile and easily broken and released in cell culture supernatants (where EVs are also found), TNTs were difficult to distinguish from EVs for their composition and function. With the goal of identifying structural components of TNTs, beside actin filaments, and possibly markers and regulators of these structures, we established a protocol of purification from U2OS cultured cells, allowing to separate TNTs from extracellular vesicles (EVs) and from cell bodies. We obtained the full composition of TNTs, compared to EVs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: MARIETTE MATONDO

LAB HEAD: Christel Brou

PROVIDER: PXD033089 | Pride | 2024-08-22

REPOSITORIES: Pride

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			Action	DRS
	20201101_Q2_TC_ColQ2-82_Brou_Mb-234T.raw	Raw
	20201101_Q2_TC_ColQ2-82_Brou_Mb-254T.raw	Raw
	20201216_Q2_TC_ColQ2-85_Brou_Mb-254-3-1.raw	Raw
	20201216_Q2_TC_ColQ2-85_Brou_Mb-254-9-1.raw	Raw
	20201216_Q2_TC_ColQ2-85_Brou_Mb-254-9-2.raw	Raw

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Project description:Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (M-bM-^IM-$ 17), indicating that 2,6-DNT exposure had relatively little impact on the brain in comparison to RDX. Birds exhibiting RDX-induced seizures accumulated over 20M-CM-^W more RDX in brain tissues in comparison to non-seizing birds even within a common dose. In parallel, expression patterns were unrelated among seizing and non-seizing birds exposed to equivalent RDX doses. In birds experiencing seizures, genes related to neuronal electrophysiology and signal transduction were significantly affected. Comparative toxicology revealed strong similarity in acute exposure effects between RDX and the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) regarding both molecular mechanisms and putative mode of action. In a manner similar to DDT, we hypothesize that RDX elicits seizures by inhibition of neuronal cell repolarization postaction potential leading to heightened neuronal excitability and seizures facilitated by multiple molecular mechanisms. Northern Bobwhite 60 Day 2,6-DNT Exposure, Brain Tissue Investigation (High Laser Intensity Scan): Juvenile male and female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 5, 10, 40, or 60 mg/kg/day). Each treatment group included 12 replicates per sex. Microarray experiments were conducted in a balanced interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the 60-day 2,6-DNT exposure comparing controls and the highest affective 2,6-DNT dose (60 mg/kg/day) in males and females, with all groups including three biological replicates for a total of 24 microarrays. To broaden signal detection, each microarray was scanned at high and low laser power to resolve low-intensity spots and reduce signal saturation, respectively (Skibbe et al., 2006). This dataset represents the High Intensity Scan.

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The proteome of Tunneling Nanotubes (TNTs) in U2OS cells; comparison with Extracellular Vesicles (EVs)

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