Figure 4
Intracellular localization of recombinant CB(SC). Transient overexpression in LCLC-103H cells visualized by WFM (A-F) or OPM (G, H). A, B. Cells expressing CB(SC)-EGFP (green) were counterstained with Hoechst33342 (blue) and LysoTracker Red (red). Diffuse, vesicular, and granular EGFP fluorescence signals were found in the cytoplasm and highly enriched inside the nucleus sparing out the nucleoli as indicated by the DNA staining. The reticular and vesicular staining of the lysosomal marker adjacent to the nuclear indentation did not overlap with EGFP signals. Obj. 40×/1.30 Oil. Processing of recombinant CB(SC) and influence of the fluorescent protein marker or the total molecule size on its localization were proven by differential tagging (N- or C-terminus, respectively) as well as by means of immunocytochemistry using a N-terminal myc-epitope. C, D. Double-tagged ECFP-CB(SC)-EYFP was distributed mainly in the nucleus analogously to CB(SC), which was marked at its C-terminus only. Accumulates were found within the nucleus and adjacent to it. Fluorescence also appeared in the ring shaped midbody matrix (enlarged region in the upper right corner). Obj. 40×/0.60; processed by deconvolution. E, F. Cells coexpressing myc-CB(SC) and CB(SC)-EGFP were fixed by acetone/methanol and immunostained against myc and GFP. A tight colocalization of both was found (E and F); the constructs were found mainly in the nucleus and stained the midbody (marked by the arrowhead; see inset). Obj. 40×/0.60. G, H. Optical sections of CB(SC)-EYFP expressing cells were subjected to spatial reconstruction (G: 3D-visualisation; H: orthogonal projection). Isosurfaces obtained by arbitrary fluorescence intensity thresholds represent distinct compartments (granules: opaque; nucleus and midbody: transparent). In the given cellular state, only a weak expression in the cytoplasm is found. The main signals arise from granular inclusions within the nucleus including distinct regions inside the nucleoli (marked by dashed lines in H) as well as from enrichment in the midbody (arrow). Obj. 63×/1.32 Oil. ROI: 72 × 59 × 11 μ m3 (= 464 × 380 × 25 voxels).