Figure 2.

GFP-fused RPP interacts via its DLC-AS region with LC8 and this region enhances NLS-dependent nuclear import via an MT-dependent mechanism. (A) Cells were transfected to express the indicated GFP fusion protein alone (single transfection) or to coexpress the indicated GFP fusion protein with dsRed-LC8 (dual transfection), and were imaged 18–24 h later by CLSM. GFP-RPP_174-297 and GFP-RPP_139-297 localize to the nucleus in transfected cells, in contrast to GFP-RPP_139-174, which is diffusely distributed similar to GFP alone (single transfection), whereas dsRed-LC8 is predominantly cytoplasmic. GFP-RPP_139-174 and GFP-RPP_139-297 colocalize with dsRed-LC8 in the cytoplasm in cotransfected cells (dual transfection, middle panel), but GFP, GFP-RPP_174-297, GFP-RPP_139-174(D₁₄₃/Q₁₄₇-A) and GFP-RPP_139-297(D₁₄₃/Q₁₄₇-A) are not affected (top and bottom panels). (B) Images such as those shown (A, single transfection) were analyzed to determine the nuclear to cytoplasmic fluorescence intensity (Fn/c), showing that RPP_139-174 does not affect nuclear localization compared with GFP alone but can increase nuclear localization of RPP_174-297. This effect is inhibitable by NCZ (added 4 h before imaging) but not CytD (added 3 h before imaging). Results are from single assays representative of three or more assays and are shown as mean ± SEM for >60 different cells. (C) CLSM images of cells treated with NCZ (4 h) or CytD (3 h) before appropriate staining show disruption of MT and actin cytoskeleton, respectively. (D) Image analysis of wild-type and D₁₄₃/Q₁₄₇-A mutated GFP-RPP_139-297 show that the mutation inhibits nuclear accumulation. Results are for combined data from five assays and are shown as mean ± SEM for >350 different cells.