Fig. 5. Decreased cytosolic tubulin concentrations in mitotic cells expressing a plasma-membrane-located high-affinity tubulin-binding chimera. (A) Depiction of the plasma-membrane-targeted CD2-RB3 chimera in complex with two /ß-tubulin heterodimers. It should be noted that RB3 is not inactivated by phosphorylation during mitosis. (B) Transfected K562 cell lines harboring the indicated pMEP-CD2 chimera derivative were Cd²⁺-induced for 20 hours, directly fixed and co-stained with anti-CD2 (R-phycoerythrin) and anti--tubulin (Alexa Fluor488). A confocal section of a representative CD2-Co and CD2-RB3-expressing mitotic cell is shown (bars, 6 µm). To allow a direct comparison with data from CD2-RB3/MAP4-coexpressing cells, presented in Figs 6 and 7, the transfected DNA was mixed with pMEP-vector Co DNA at a ratio of 1:1. (C) The average fluorescence intensity of non-polymeric -tubulin in confocal sections was determined in the cytosol and at the plasma membrane. The average values of 50 analyzed cells expressing the indicated CD2 chimera is presented. (D) The -tubulin staining fluorescence intensities of individual cells expressing the indicated CD2 chimera are plotted. All data in this figure are derived from the same transfected cell populations but are representative of at least three independent transfection experiments.