BiFC-Assay

The BiFC (Bimolecular Fluorescence Complementation) assay is based on the observation that N- and C-terminal subfragments of GFP (or derivatives thereof, e.g.

YFP) do not spontaneously reconstitute a functional fluorophore. However, if fused to interacting proteins, the two non-functional halves of the fluorophore are brought into tight contact, refold together and generate de novo fluorescence. Thus, by BiFC, the interaction status of two proteins of interest can be easily monitored via fluorescence emission upon excitation with a suitable wavelength. A schematic image of the principle of the BiFC assay is depicted in figure 3.28.

Figure 3.28: Principle of the BiFC assay. The scheme depicts the principle of the BiFC assay, exemplified by a split YFP fluorophore. Proteins A and B are fused to N- and C-terminal fragments of YFP, respectively. In the absence of an interaction between A and B, the fluorophore halves remain non-functional. Following interaction between A and B, a functional fluorophore is reconstituted which exhibits emission of fluorescence upon excitation with an appropriate wavelength (figure taken from Bhat, 2006).

The BiFC-Assay used in this study was established by Prof. Dr. S. Hoyer-Fender (Department of Developmental Biology, University of Göttingen). The vectors FPCA-V1 and FPCA-V2 were generated and kindly provided by Prof. Dr. S. Hoyer-Fender.

The modified vector FPCA-V1 contains the constitutive CMV promoter, followed by the ORF of the putative LIS1 interacting proteins (ACT, BRAP and NUDEL, respectively), a short spacer region and the C-terminal end of EGFP (amino acids 158 to 239). The modified vector FPCA-V2 contains the constitutive CMV promoter, followed by E2 tag fused to the N-terminal end of EGFP (amino acids 1 to 157), a short spacer region and the ORF of Lis1 (Fig. 3.29).

Figure 3.29: Schematic drawing of the vectors used for BiFC-Assay (provided by Prof. Dr. S. Hoyer-Fender). In FPCA-V1-iP ORFs of the putative interaction partners (iP) of LIS1 were cloned 5`upstream

of the C-terminal part of EGFP (amino acids 158-239). ORF of Lis1 was cloned 3`downstream of the N-terminal part of EGFP (amino acids 1-157) in FPCA-V2-Lis1.

The ORFs for all constructs were amplified with primers described in 2.1.8, cloned into pGEM-T Easy, sequenced and subcloned into FPCA-V1 and FPCA-V2, respectively.

3.2.2.1 Interaction of LIS1 and LIM-only-protein-ACT

NIH-3T3 cells and HeLa cells were transiently transfected with FPCA-V1-LIM-only-protein-ACT and FPCA-V2-Lis1. After 48 hrs cells were fixed, counterstained with DAPI and examined under the confocal microscope IX81 (Olympus). EGFP fluorescence could be detected in cytoplasm of NIH-3T3 cells (Fig. 3.30 A, B) as well as in cytoplasm of HeLa cells (Fig. 30, C). As a positive control, cells were transfected with FPCA-V1-Pelota and FPCA-V2-CDK2-AP1 (Fig. 3.30 D). Interaction of these proteins has been shown by O. Burnicka-Turek (unpublished data). To check the specificity of the vectors, NIH-3T3 cells were cotransfected with FPCA-V1-LIM-only-protein-ACT and FPCA-V2-CDK2-AP1 (Fig. 3.30 E), and FPCA-V1-Pelota and FPCA-V2-Lis1, respectively (Fig. 3.30 F). No EGFP fluorescence could be detected in these negative controls.

Figure 3.30: Confocal images of specific interaction of LIS1 and Lim-only protein-Act detected by BiFC-Assay. NIH-3T3 (A, B) and HeLa (C-F) cells were transiently transfected with FPCA-vectors, fixed 48h after transfection and counterstained with DAPI. Interaction of LIS1 and Lim-only-protein-Act is clearly visible in NIH-3T3 cells (A,B) and in HeLa cells (C). As a positive control the interaction of Pelota and CDK2-AP1 is shown in D. Specificity of the vectors was analysed by transfection of cells with FPCA-V1-Lim-only-protein-Act and CDK2-AP1 (E) and FPCA-V1-Pelota and FPCA-V2-Lis1 (F), respectively. No EGFP fluorescence was detected in these negative controls. All pictures are dual image overlays of EGFP and DAPI fluorescences.

3.2.2.2 Interaction of LIS1 and BRCA1-Associated-Protein

HeLa cells were transiently transfected with FPCA-V1-BRAP and FPCA-V2-Lis1.

After 48 h, cells were fixed, counterstained with DAPI and examined under the confocal microscope IX81 (Olympus). EGFP fluorescence could be detected in cytoplasm of HeLa cells (Fig. 3.31 A, B). To check the specificity of the vectors, NIH-3T3 cells were cotransfected with FPCA-V1-BRAP and FPCA-V2-CDK2-AP1 (Fig. 3.31 C), and FPCA-V1-Pelota and FPCA-V2-Lis1, respectively (Fig. 3.31 D). No EGFP fluorescence could be detected in these negative controls. The experiment was repeated twice to confirm the interaction between LIS1 and BRAP.

Figure 3.31: Confocal images of specific interaction of LIS1 and BRAP detected by BiFC-Assay. HeLa cells were transiently transfected with FPCA-vectors, fixed 48h after transfection and counterstained with DAPI. Interaction of LIS1 and BRAP is clearly visible in cytoplasm of HeLa cells (A, B). No EGFP fluorescence was detected in negative controls (C, D). HeLa cells were transiently transfected with FPCA-V1-BRAP and FPCA-V2-CDK2-AP1 (C) and FPCA-V1-Pelota and FPCA-V2-Lis1 (D), respectively. All pictures are dual image overlays of EGFP and DAPI fluorescences.

3.2.2.3 Interaction of LIS1 and NUDEL

HeLa cells were transiently transfected with FPCA-V1-Nudel and FPCA-V2-Lis1.

After 48 hrs cells were fixed, counterstained with DAPI and examined under the confocal microscope IX81 (Olympus). EGFP fluorescence could be detected in cytoplasm of HeLa cells (Fig. 3.32 A, B). Nudel (Nuclear distribution gene E-like) is involved in neuronal migration during brain development. To check the specificity of the vectors, NIH-3T3 cells were cotransfected with FPCA-V1-Nudel and FPCA-V2-CDK2-AP1 (Fig. 3.32 D). EGFP fluorescence could be detected in the negative control, while in untransfected HeLa cells no EGFP fluorescence was detected (Fig. 3.32 C).

The experiment was repeated twice to confirm the interaction between LIS1 and NUDEL.

Figure 3.32: Confocal images of specific interaction of LIS1 and NUDEL detected by BiFC-Assay.

HeLa cells were transiently transfected with FPCA-vectors, fixed 48h after transfection and counterstained with DAPI. Interaction of LIS1 and NUDEL is clearly visible in cytoplasm of HeLa cells (A, B). No EGFP fluorescence was detected in untransfected HeLa cells (C), but transiently transfected cells with FPCA-V1-NudeL and FPCA-V2-CDK2-AP1 show a clear EGFP signal (D). All pictures are dual image overlays of EGFP and DAPI fluorescences.