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Molecular and Cellular Biology, December 2001, p. 8615-8625, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8615-8625.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

Hyewon Phee,1,2 William Rodgers,3 and K. Mark Coggeshall1,4,*

Immunobiology and Cancer1 and Molecular Immunogenetics3 Programs, The Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, and Departments of Biochemistry2 and Microbiology,4 The Ohio State University, Columbus, Ohio 43210

Received 26 June 2001/Accepted 13 September 2001

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (Fcgamma RII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that Fcgamma RII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in Fcgamma RII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were corecruited to the BCR, regardless of the presence or absence of Fcgamma RII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of Fcgamma RII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/Fcgamma RII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

* Corresponding author. Mailing address: The Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, 825 N.E. 13th. St., Oklahoma City, OK 73104. Phone: (405) 271-7905. Fax: (405) 271-8568. E-mail: mark-coggeshall{at}omrf.ouhsc.edu.

Molecular and Cellular Biology, December 2001, p. 8615-8625, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8615-8625.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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