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LC-MS of FAPS-sorted ELVAs from mouse oocytes

ABSTRACT: We discovered that mouse oocytes contain novel non-membrane-bound organelles which we named EndoLysosomal Vesicular Assemblies (ELVAs). ELVAs contain endolysosomal and autophagy vesicles as well as Proteasomes held together by a matrix protein. To discover the matrix protein holding ELVAs together we subjected mouse oocyte with fluorescently labelled ELVAs to mechanical lysis followed by Fluorescence-Activated Particle Sorting to enrich for ELVAs. Enriched ELVAs were then subjected to proteomics.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Oocyte

SUBMITTER: Gabriele Zaffagnini

LAB HEAD: Elvan Böke

PROVIDER: PXD042768 | Pride | 2024-02-20

REPOSITORIES: Pride

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			Action	DRS
	2020MQ022_GAZA_001_02_45pto.pdResult	Other
	2020MQ022_GAZA_001_02_45pto.raw	Raw
	2020MQ022_GAZA_002_02_45pto.pdResult	Other
	2020MQ022_GAZA_002_02_45pto.raw	Raw
	2020MQ028_GAZA_001_01_90pto.pdResult	Other

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Mass Spectrometry of FAPS sorted ELVAs by an alternative sorting strategy

Project description:We discovered that mouse oocytes contain novel non-membrane-bound organelles which we named EndoLysosomal Vesicular Assemblies (ELVAs). ELVAs contain endolysosomal and autophagy vesicles as well as Proteasomes held together by a matrix protein. To discover the matrix protein holding ELVAs together we subjected mouse oocyte with fluorescently labelled ELVAs to mechanical lysis followed by Fluorescence-Activated Particle Sorting to enrich for ELVAs. Enriched ELVAs were then subjected to proteomics.

2024-02-20 | PXD047716 | Pride

LC-MS of control vs BafA1-treated mouse oocytes during maturation

Project description:We discovered that mouse oocytes contain novel non-membrane-bound organelles, which we named EndoLysosomal Vesicular Assemblies (ELVAs). ELVAs contain endolysosomal and autophagy vesicles, as well as proteasomes. In immature oocytes, ELVAs store ubiquitinated proteins and show little degradative activity. During oocyte maturation, ELVAs increase protein degradation, thereby degrading the stored proteins. To understand which proteins are being degraded inside ELVAs during oocyte maturation we treated mouse oocytes with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) during maturation and subjected equal number of in vitro matured oocytes to mass spec, looking for enriched proteins in the BafA1-treated sample.

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Autophagy cargo receptors are secreted via extracellular vesicles and particles in response to endolysosomal inhibition or impaired autophagosome maturation

Project description:The endosome-lysosome (endolysosome) system plays central roles in both autophagic degradation and secretory pathways, including the exocytic release of extracellular vesicles and particles (EVPs). Although previous work has revealed important interconnections between autophagy and EVP-mediated secretion, our molecular understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition that mediates the EVP-associated extracellular release of autophagic cargo receptors, including p62/SQSTM1. This extracellular secretion is highly regulated and critically dependent on multiple ATGs required for the progressive steps of early autophagosome formation as well as Rab27a-dependent exocytosis. Furthermore, the disruption of autophagosome maturation, either due to genetic inhibition of the autophagosome-to-autolyosome fusion machinery or blockade via the SARS-CoV2 viral protein ORF3a, is sufficient to induce robust EVP-associated secretion of autophagy cargo receptors. Finally, we demonstrate that this ATG-dependent, EVP-mediated secretion pathway buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Based on these results, we propose that secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis in response to endolysosomal dysfunction or impaired autophagosome maturation.

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Impact of cellular miRNAs on the biomarker signatures of circulating miRNAs

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Analysis of the impact of cellular miRNA signatures on the profiles of circulating miRNA biomarkers (fractionation data)

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