Figure 1. Multi-color translocation biosensor assays to analyse Taspase1-mediated cleavage in living cells.

A. In vivo assay. Schematic domain organization and principle of the TS-Cl2⁺ translocation sensor to probe Taspase1 activity. TS-Cl2⁺ is composed of GST, GFP, combinations of a nuclear import (NLS) and a Myc epitope-tagged export (NES) signal, combined with the Taspase1 cleavage site from MLL (Cl2⁺; aa ²⁷¹³KISQLDGVDD²⁷²²). TS-Cl2⁺ localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and the cytoplasm. Co-expression of Taspase1-BFP (Tasp-BFP) results in the loss of the NES by proteolytic cleavage of the biosensor, triggering nuclear accumulation of the green fluorescent indicator protein. B. Expression of Taspase1 results in nuclear translocation of TS-Cl2⁺ in living HeLa transfectants. The autofluorescent biosensor is predominantly cytoplasmic (upper panel), whereas co-expression of Taspase1-BFP results in its proteolytic cleavage and nuclear accumulation (lower panel). Myc-NES_Rev was detected by indirect immunofluorescence using an α-myc-tag Ab. C. The translocation biosensor is functional not only in adherent but also in leukemia cell models. K562 cells were transfected with expression plasmids encoding the indicated proteins. Coexpression of Tasp-GFP but not of inactive Tasp^T234V-GFP resulted in proteolytic cleavage and nuclear accumulation of the red fluorescent biosensor, TS-Cl2⁺_R. Biosensor localization was analyzed 48 h post transfection in at least 200 fluorescent living cells, and representative images are shown. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. D. Quantitation of TS-Cl2⁺ processing in vivo. HeLa cells were transfected with the indicated ratios of enzyme (Taspase1-BFP) and substrate (TS-Cl2⁺). 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 200 fluorescent cells. Cleavage-induced nuclear accumulation of the biosensor significantly increased already at a ratio of 1/10 (***: p<0.0001). Results from a representative experiment are shown. E. Biosensor assay specificity. Biosensors containing a non-functional Taspase1 cleavage site (TS-Cl2⁺_mut) or a cleavage site for Caspase3 (BS-Casp3) remained cytoplasmic upon co-expression of Tasp-BFP. No nuclear accumulation of TS-Cl2⁺ was observed upon coexpression of inactive Tasp^T234V-BFP or the nucleolar RevM10BL-BFP protein. F. Cleavage of TS-Cl2⁺, TS-Cl2⁺_mut or BS-Casp3 analyzed by immunoblot. 293T cells were transfected with the indicated biosensors together with the indicated Taspase1 expression plasmids, the empty vector (control), RevM10BL-BFP or the protease Caspase3. Expression of proteins and cleavage products in cell lysates was visualized using α-GST, -Taspase1, -GFP or -Casp3 Abs. GAPDH served as loading control. G. Ectopic expression of Caspase3 does not induce cleavage and nuclear translocation of TS-Cl2⁺. Caspase3 expression was visualized by IF using α-Casp3 antibody, its activation by α-ClevCasp3 Ab. GFP/BFP were visualized by fluorescence microscopy. Scale bars, 10 µm.