Study on intracellular trafficking of Mac-1 by direct visualization.
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 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.
Yan
Ming
M
Department of Pathophysiology, Second Military Medical University, Shanghai 200433, China. myan00@hotmail.com
Mao
Jifang
J
Wei
Yi
Y
Zhong
Jigen
J
Yang
Shengsheng
S
Xu
Renbao
R
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D006224:Cricetinae; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000235:genetics / Q000378:metabolism*; D008280:Magnetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D008164:Luminescent Proteins; D016177:Macrophage-1 Antigen; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D018799:Intercellular Adhesion Molecule-1; D013755:Tetradecanoylphorbol Acetate