The Role Of Rab6in The Phagocvtosis Of Mouse Macrophage RAW264.7Cell Line

Effective phagocytosis results from particle internalization and phagosomal maturation. After engulfment by macrophages, the microorganisms are trapped, together with extracellular fluid, in a vacuole, or phagosome, derived from the plasma membrane. Because the nascent phagosomal membrane and its contents are innocuous, they must undergo a drastic conversion to acquire the microbicidal and degradative features associated with innate immunity. This conversion, known as phagosomal maturation, is accomplished through a strictly choreographed sequence of fusion and fission events that involve defined compartments of the endocytic pathway.Rab GTPases can interact with upstream regulatory factors and specific downstream effectors and act as the molecular switch of vesicle trafficking. Rab proteins play important roles in the formation, transport, adhesion, anchoring and fusion process of vesicles. Many Rab proteins have been found to be involved in cell endocytic pathway. Based on the previous studies in our lab, it is found that the Rab6protein, a member of Rab superfamily, is involved in the antiviral phagocytosis of invertebrates. Therefore the mouse macrophage cell line, RAW264.7, was used in this study to reveal the role of Rab6in the phagocytosis regulation of mammalian cells.To explore the function of Rab protein in the phagocytosis of RAW264.7, the Rab6gene was silenced and rescued, respectively. We found no significant change in the phagocytic activity of Rab6-koncked-down RAW264.7cells using FITC-labeled inactivated E.coli, while the degradation ability of engulfed bacteria was weakened. The pHrodoTM-labeled E.coli phagocytic assays and FITC-labeled E.coli co-localization with lysosomes showed that the Rab6protein significantly affected acidification of the phagosome. When the Rab6gene was rescued by transfection of the recombinant construct containing the synonymous mutations of Rab6gene, the mean fluorescence intensity of pHrodoTM-labeled E.coli phagocytosis and lysosomal co-localization of FITC-labeled E.coli were both enhanced. The results indicated that the Rab6protein participated in the regulation of phagosomal maturation process of RAW264.7macrophage.The intracellular protein expression levels of Rab5, Rab6and Rab7were decreased by specific siRNAs. The results showed that Rab6-knocked-down treatment shared similar data with that of Rab7-knocked-down cells. The findings indicated that Rab6might regulate the midanaphase of phagosomal maturation. By means of the real-time observation of living cells, it was found that the Rab6protein aggregated from Golgi to the phagosome after the E.coli was engulfed. It indicated that Rab6might be involved in the transportation of vesicles between the Golgi and phagosome, thus regulating the maturation process of phagosome.BICD1is one of the most common effector proteins of Rab6. The results showed that the knockdown of BICD1exhibited consistent data with those of Rab6-knocked-down cells, indicating that BICD1protein was also involved in phagosomal maturation process, and most likely jointly exercised the same function with Rab6protein. Rab6was interacted with BICD1, and mediated the vesicular transportation along microtubules between the Golgi apparatus and phagosome. When the expression levels of Rab6and BICD1were down-regulated simultaneously, the data presented that the phagocytic activity and phagosomal acidification of RAW264.7cells were significantly decreased. The date suggested that intracellular membrane system plays an important role in cell phagocytosis, which was helpful to understand the process of phagocytosis in depth.