Abstract
Previously, we constructed DNA vectors containing cDNAof Mac-1 subunits (CD11b or CD18b) fused with fluorescence protein (FP). cDNA fragments and the DNA constructs were then transfected into CHO cells (as CHO-Mac-1-FP). The structure and function of Mac-1-FP obtained from the CHO-Mac-1-FP cells are nearly identical to that expressed in wild type leukocytes. In the present study, the intracellular trafficking of Mac-1 was visualized directly by monitoring the fluorescent intensities of YFP-CD18 and PE-conjugated monoclonal antibody against CD11b under a confocal microscope in CHO-Mac-1-FP cells. The results indicate that: (i) although Mac-1 was not detected in the cell membrane at resting state, it had been translocated and clustered into the cell membrane by 1 h and internalized 2 h after PMA stimulation, at which point the fluorescence intensity began to diminish gradually, probably due to partial degradation of Mac-1. The fluorescence of CD18 and CD11b reappeared on the cell membrane 1 h after re-treatment with PMA, suggesting the recycling of non-degraded Mac-1. (ii) The adhesion rate of CHO-Mac-1-FP to magnetic beads coupled ICAM-1 increased within 4 h after their initial interaction, accompanied by the clustering of Mac-1-FP. After 8 h, the adhesion rate declined and fluorescence also decreased simultaneously. The pattern of change in fluorescence in CHO-Mac-1-FP cells elicited by ICAM-1 beads was similar to that elicited by PMA, suggesting that endocytosis and degradation of Mac-1 occurred after the interaction with ICAM-1. Thus, we conclude that the intracellular trafficking of Mac-1 after activation is associated with membrane translocation, endocytosis, degradation and recycling. These changes are in parallel with the adhesion of CHO-Mac-1-FP cells with ICAM-1, and may be involved in the adhesion and detachment of leukocytes. The detachment of leukocytes may be caused by endocytosis of Mac-1.
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Yan, M., Mao, J., Wei, Y. et al. Study on intracellular trafficking of Mac-1 by direct visualization. Sci. China Ser. C.-Life Sci. 47, 521–529 (2004). https://doi.org/10.1360/02yc0232
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DOI: https://doi.org/10.1360/02yc0232