| This thesis focuses on two aspects of compartmentalization with respect to BCR signaling.{09}In the first section, compartmentalization of the BCR to lipid rafts is considered and in the second section the subsequent compartmentalization of BLNK and PLCgamma to the BCR is considered. Recent studies have suggested that the BCR translocates into lipid rafts in certain B cell stages. Yet these reports have been ambiguous and the mechanisms regulating such translocation have remained elusive. In this thesis it is demonstrated that the BCR can translocate into lipid rafts following BCR cross-linking in the immature B cell lines, WEHI 231 and CH31 (Jackson et al., 2005). Additionally, it is shown that the Igalpha/beta heterodimer, in the absence of the mIgM subunit, can translocate into lipid rafts in the immature B cell line, WEHI 303.1.5 (Jackson et al., 2005). Previous studies have likewise demonstrated that the mIgM subunit, in the absence of Igalpha/beta, can translocate into lipid rafts (Cheng et al., 2001). Together, these findings may be used to help define a structural feature, common to both subunits, involved in mediating lipid raft association.; The PLCgamma pathway is an integral part of the BCR signaling network. Loss-of-function studies have indicated that the BCR is coupled to PLCgamma via Syk, BTK and BLNK. In this thesis, a non-lymphoid reconstitution system was used to determine if these components are sufficient to couple the BCR to PLCgamma. From this it was determined that co-expression of the BCR, Syk and BLNK is sufficient to reconstitute BCR-induced PLCgamma activation in the system. However, this activation is hypothesized to represent only a partial reconstitution of the pathway as neither BLNK nor PLCgamma are recruited to the plasma membrane upon BCR cross-linking and as PLCgamma phosphorylation appears very limited. It was hypothesized that this might be due to the absence of BTK; however, further expression of BTK within the system inhibited rather than enhanced PLCgamma activation. Subsequent investigations determined that BTK is constitutively activated within this system and as such, may be inappropriately affecting the pathway. Additionally, it was hypothesized that the limited reconstitution may be a consequence of the inability to reconstitute BCR-induced BLNK and PLCgamma membrane recruitment. Thus, BLNK and PLCgamma were constitutively targeted to the plasma membrane within the system. From this, it was determined that membrane-targeting of PLCgamma is sufficient to reconstitute BCR-induced, Syk-dependent PLCgamma activation. In contrast, membrane-targeting of BLNK is not sufficient to reconstitute BCR-induced PLCgamma membrane recruitment or to enhance BCR-induced PLCgamma activation within this system. This suggests that there may be an additional defect in the system that is preventing the formation of a functional BCR/BLNK/PLCgamma signaling complex. Moreover, these findings suggest that there may be a deficit in our current understanding of the BCR/PLCgamma pathway.; In summary, these findings highlight the importance of compartmentalization in BCR signaling both with respect to compartmentalization of the BCR to lipid rafts and the subsequent compartmentalization of BLNK and PLCgamma to the BCR. |
