Fig. 1. Differential anchorage probes (DAPs) targeting γ-secretase activity. (A) APP and its β-secretase derived fragment, C99, were tagged with GFP at their C-terminal domain. The action of γ-secretase on C99-GFP (as well as on APP-GFP after β-secretase cutting), allows the release of Aβ in the extracellular space (or within the lumen of intracellular compartments) and AICD-GFP fragment in the cytosol. (B) Schematic representation of cell fluorescence before and after plasma membrane permeabilization (PMP) in cells with functional or inhibited γ-secretase. Only membranous fluorescence is conserved upon PMP: by comparing residual membrane-bound fluorescence with the total one, measured before permeabilization, the fluorescence retention ratio is determined, as an inverse index of γ-secretase activity.

Fig. 2. Characterization of the new GFP probes cutting pattern in HeLa (A) and SH-SY5Y cells (B). Cells were transiently transfected with the GFP probe, and treated overnight with DAPT (1 μM), L-685,458 (1 μM), z-VAD (50 μM) or a combination of these drugs. Protein extracts were separated by electrophoresis, blotted and analyzed with an anti-GFP antibody. The untreated samples (NT) reveal an efficient cleavage of the probe by γ-secretase in both cell models, as demonstrated by the presence of the 35 kDa AICD-GFP band. See Results for details. (C) Protein extract from HeLa cells transfected with the mutated C99-GFP construct (mC99-GFP), lacking the consensus sequence for caspases cleavage, analyzed as in panel A. (D) Western blotting of APP-GFP transfected HeLa cells. A band corresponding to the full-length protein (APP-GFP, 137 kDa) as well as the C83-GFP, AICD-GFP and free GFP bands are shown in the untreated sample (NT). See Results for details.

Fig. 3. Differential distribution of mC99-GFP in cellular compartments upon γ-secretase activity. HeLa cells were transfected with mC99-GFP, treated overnight, or not, with DAPT (1 μM) and L-685,458 (1 μM) and then permeabilized with digitonin (50 μM, 3 min); the soluble and membranous fractions were collected separately. Total (Tot), soluble (S) or membranous (M) protein fractions were analyzed by means of Western blot with an anti-GFP antibody. Note that cell treatment with DAPT and L-685,458 changes the overall distribution of the probe, indicating that our probe works properly (see Results for detailed description).

Fig. 4. Confocal images of HeLa cells expressing either mC99-GFP or APP-GFP. (A) Living HeLa cells transiently transfected with mC99-GFP were observed 4 h after transfection and followed up to 2 h. Fluorescence images were acquired every 360 s (from the first image, on the top-left of the panel, to the last one, on the bottom-right). Note the increase in cytosolic fluorescence, indicating AICD-GFP release in the cytosol upon γ-secretase activity. (B) HeLa cells transiently transfected with mC99-GFP were observed after 4 h from transfection and permeabilized by digitonin. Fluorescence images were acquired every 12 s (from the first image, on the top-left of the panel, to the last one, on the bottom-right). After few minutes all the cytosolic fluorescence is lost and a membranous staining is obtained. (C) mC99-GFP stably-expressing HeLa cells treated (on the right) or not (on the left) with DAPT (1 μM, overnight). (D) APP-GFP transiently transfected HeLa cells were observed 4 h after transfection and followed up to 1 h. Fluorescence images were acquired every 240 s (from the first image, on the top-left of the panel, to the last one, on the bottom-right) and an increasing cytosolic fluorescence during time is disclosed. Bars, 10 μM.

Fig. 5. Cytometric measurement of γ-secretase activity.(A) C99-GFP or mC99-GFP transiently transfected HeLa cells were treated overnight with DAPT (1 μM), L-685,458 (1 μM), z-VAD (50 μM) or a combination of these drugs. The membrane retention ratio, calculated as the number of cells with membrane-bound fluorescence after cell permeabilization, over the total fluorescent cell number before digitonin treatment, is shown as an inverse index of γ-secretase activity. (B) γ-Secretase activity detected in mC99-GFP stable expressing HeLa cells treated as in panel A. Note that inhibition of γ-secretase by DAPT or L-685,458 is almost complete in this stable cell line, as shown by a retention values over 80%. (C) Cytometric measurement of γ-secretase activity in APP-GFP transiently transfected HeLa cells. DAPT and L-685,458 treatments (as in panel A) induce a strong increase in probe membrane retention, indicating the correct behaviour of our probes. n ≥ 3; p < 0.05; p < 0.001.

Fig. 6. Effect of γ-secretase and caspase inhibitors on GFP retention ratio and apoptosis. (A) HeLa cells transfected with GFP were treated overnight, or not, with DAPT (1 μM), L-685,458 (1 μM) and z-VAD (50 μM) and tested for membrane fluorescence retention. n ≥ 6. (B) HeLa cells transfected with mC99-GFP were treated overnight with the indicated γ-secretase inhibitor concentrations. Apoptotic cells were counted as the number of cells lacking TMRM fluorescence over total cells in the FL3 channel (530 nm). n = 3.

Fig. 7. Cytometric measurement of DAPT and L-685,458 half maximal inhibitory concentration (IC₅₀) for γ-secretase activity in stable expressing mC99-GFP HeLa cells. HeLa cells were treated overnight with the indicated drug concentration and analyzed as described in Fig. 5. For DAPT and L-685,458 an IC₅₀ of 66 nM and 57 nM, respectively, were determined. For each drug concentration, n = 3.

Fig. 8. Effect of specific inhibitors and FAD-linked PSs expression on γ-secretase activity in MEF cells. (A) MEFs wt or knocked-out for PS1 and PS2 (MEF KO) were transiently transfected with mC99-GFP, treated overnight with DAPT (1 μM) or L-685,458 (1 μM) and tested for fluorescence retention. Note that in MEFs KO there is no effect of γ-secretase inhibitors on probe retention ratio, which is already very high in the control condition. (B) Modulation of γ-secretase activity by PSs expression. MEFs wt or KO were transiently transfected with mC99-GFP and the indicated PS and analyzed after 24 h. Note that in MEF KO FAD-PS mutations failed to recover γ-secretase activity, acting as loss of function mutations. (C, D) MEF KO cells were transiently transfected with mC99-GFP together with one of the above reported PS forms and the cytometric assay was carried out after 24 h. n ≥ 5; p < 0.05; p < 0.001.

Fig. 9. Cytometric measurement of γ-secretase activity on NotchΔE-GFP: effect of inhibitors and PS expression in HeLa, SH-SY5Y and MEF KO cells. Cells, transiently transfected with the NotchΔE-GFP construct, were treated as described in Fig. 8. n ≥ 3; p < 0.05; p < 0.001.