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Study on intracellular trafficking of Mac-1 by direct visualization

Science China(Life Sciences) 2004年第6期47卷 521-529页

作者： YAN Ming1, MAO Jifang2, WEI Yi3, ZHONG Jigen1, YANG Shengsheng2 & XU Renbao1 1. Department of Pathophysiology, Second Military Medical University, Shanghai 200433, China 2. Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai 200433, China 3. Biological Science School, Fudan University, Shanghai 200433, China Correspondence should be addressed to Yan Ming 1. Department of Pathophysiology Second Military Medical University 200433 Shanghai China 2. Department of Biochemistry and Molecular Biology Second Military Medical University 200433 Shanghai China 3. Biological Science School Fudan University 200433 Shanghai China

Previously, we constructed DNA vectors containing cDNA of 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 leuko- cytes. In the present study, the intracellular trafficking of Mac-1 was visualized directly by moni- toring 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) al- though 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 in- teraction, 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 de- tachment of leukocytes. The detachment of leukocytes may be caused by endocytosis of Mac-1.

关键词： Mac-1 (macrophage differentiation antigen associated with complement three receptor function), pma, ICAM-1, trafficking.

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