Dataset Information

Pacific oyster phagosome LC-MSMS

ABSTRACT: Phagosomes are task-force organelles of innate immune systems responsible for the recognition, processing, and ultimately destruction of invading microorganisms. Among invertebrate and vertebrate species, evolutionary diversity and continuity abound in the protein machinery executing this coordinately regulated process, though its dynamics and full scope of regulators remain incompletely understood. In order to clarify molecular mechanisms underlying phagocytosis, we studied phagocyte response to beads and Vibrio species, using hemocytes of the Pacific oysters (Crassostrea gigas) as a marine invertebrate model. Phagosomes from different stages of phagocytosis were isolated by density-gradient centrifugation, and more than 400 phagosome-associated proteins were subsequently identified via high-throughput quantitative proteomics. In modeling key networks of phagosomal protein-protein interactions, our results support the essential roles of several processes driving phagosome formation and maturation, including actin cytoskeleton remodeling, and signal transduction by myosin and Rab proteins. In further quantitative proteomic analysis, trends of both upregulation and downregulation of protein expression were observed proteins during phagosome formation and maturation. Notably, the signal transducers CgRhoGDI and CgPI4K were implicated. In addition, six Rab proteins including Rab1, Rab2, Rab7, Rab11, RaB21, and Rab33 were also identified as active regulators or mediators in hemocyte phagocytosis. Our current work illustrates the diversity and dynamic interplay of phagosomal proteins, providing a framework for better understanding host-microbe interactions during phagosome formation and maturation in under-examined invertebrate species.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Crassostrea Gigas (pacific Oyster) (crassostrea Angulata)

TISSUE(S): Blood Cell, Hemocyte

SUBMITTER: Huawei MU

LAB HEAD: Ziniu Yu

PROVIDER: PXD015810 | Pride | 2020-04-27

REPOSITORIES: Pride

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Publications

Hemocyte phagosomal proteome is dynamically shaped by cytoskeleton remodeling and interorganellar communication with endoplasmic reticulum during phagocytosis in a marine invertebrate, Crassostrea gigas.

Mao Fan F Mu Huawei H Wong Nai-Kei NK Liu Kunna K Song Jingchen J Qiu Jianwen J Lin Yue Y Zhang Xiangyu X Xu Duo D Xiang Zhiming Z Li Jun J Zhang Yang Y Yu Ziniu Z

Scientific reports 20200420 1

Phagosomes are task-force organelles of innate immune systems, and evolutionary diversity and continuity abound in the protein machinery executing this coordinately regulated process. In order to clarify molecular mechanisms underlying phagocytosis, we studied phagocyte response to beads and Vibrio species, using hemocytes of the Pacific oysters (Crassostrea gigas) as a marine invertebrate model. Phagosomes from different stages of phagocytosis were isolated by density-gradient centrifugation, a ...[more]

PMID: 32313134

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Project description:The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions. All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 Âµm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record. http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

			Action	DRS
	LP1-3EP1-3_1.msf	Msf
	LP1-3EP1-3_1.raw	Raw
	LP1-3EP1-3_2.msf	Msf
	LP1-3EP1-3_2.raw	Raw
	LP1-3amp3EP1-3_1.raw.mgf	Mgf