Figure 7
Localization of mutated CB-FP chimaeras in transiently expressing LCLC-103H cells. Individual expression with corresponding constructs (above: EYFP-tagged; below: ECFP-tagged) and false colour superposition of both fluorescence channels (YFP: red; CFP: green) are shown. Double-transfection of Δ72CB (A) or CB([C211_I243del]SC) (B) with CB(SC), respectively, resulted in a complete colocalization of both constructs. The heavy chain domain still colocalized with the single form to a large extent (C). Compared with the heavy chain, the light chain of CB was distributed homogeneously throughout the cell as pure GFP (D). In contrast to the CB(HCN') constructs, CB(HCC') expressed hotspots around the Golgi and inside the nucleus and stained the midbody (E). Transient coexpression of CB(SC) and its increasingly truncated C-terminal fragments (F-J) revealed decreased nuclear fluorescence with smaller fragment size. Already in case of CB(HCC'1) (F), the tendency to accumulate was reduced considerably and the accumulations disappeared completely in case of CB(HCC'4) transfection (I). The latter showed an almost complete homogeneous signal distribution without any accumulation in the nucleus, the granules, or the midbody, respectively. The elimination of 9 residues in the C-terminal part of CB (Q268_G277del) had no significant influence on the localization; the construct still colocalized completely with CB(SC) (J; the inset is a superposition of the midbody fluorescence with the corresponding phase contrast image). A-C, G-I. OPM; λEx.(ECFP): 430 nm; λEx.(EYFP): 500 nm; obj. 60×/1.2 Plan Apo water. D-F, J. WFM, in part after deconvolution (D, J); obj. 63×/1.25 Oil (D, F, J); obj. 40×/0.60 (E); bars 10 μ m.