Fig 1.
Schematic of fluorescent dye Rhodamine 110 (Rh110) activation following proteolytic cleavage by effector Separase.
Rh110 contains two amino groups allowing for bi-conjugation to the C-terminal end of the Separase-specific substrate peptide ([Cy5-D-R-E-I-M-R]2-Rh110). When in this form, the molecule is essentially non-fluorescent. Active Separase cleaves the amide bonds between the peptides and the rhodamine. The Rh110 becomes fluorescently active and this increase in fluorescence can be measured by flow cytometry or fluorescence spectrophotometry (Exmax 488 nm, Emmax 535 nm). Aminoterminal conjugation of Cy5 serves for monitoring peptide uptake by target cells as internal standard (Exmax 635 nm, Emmax 650 nm). The location of amino- and carboxy-termini of the conjugated peptides are shown by the letters N and C, respectively.
Fig 2.
Kinetics and flow cytometric detection of [Cy5-D-R-E-I-M-R]2-Rh110 peptide uptake (Cy5 fluorescence) and peptide cleavage (Rh110 fluorescence) in U937 cells.
(A) U937 cells were incubated with 10 μM peptide in complete RPMI-1640 medium for increasing time periods (1–240 min) followed by flow cytometric analysis according to the standard protocol as described in Materials & Methods. (B) Visualization of peptide uptake by Cy5 fluorescence microscopy. Equal amounts of peptide-treated (10μM, 90 min) and untreated cells were mixed and subjected to fluorescence microscopic analysis. Cy5 fluorescence is shown in pink, nuclei are counterstained with DAPI (blue). (C) Time kinetics of peptide uptake was calculated as the percentage of peptide (Cy5) positive cells (triangular dots) and the mean fluorescence per cell (round dots). (D) Time kinetics of Separase-related peptide cleavage was calculated as the percentage of Rh110 positive cells (triangular dots) and the mean fluorescence per cell (round dots). (E) Peptide dose (0–20 μM) dependency of Separase-related peptide cleavage. In all experiments, peptide alone (Ac-D-R-E-I-M-R) without dye (Cy5, Rh110) conjugation was used as non-fluorogenic control substrate and cellular autofluorescence gating control. Abbreviations: RFU, relative fluorescence units; fluor., fluorescence; conc., concentration; min, minutes.
Fig 3.
Separase activity during mitotic progression in U937 cells.
(A) U937 cells arrested in G2/M by nocodazole were monitored by flow cytometry at 0, 90, 180, and 270 min after release from the nocodazole block. (B) Influence of FCS supplementation on Separase activity and mitotic progression. After serum starvation cells were incubated with increasing percentages (10%, 50%, 100%) of FCS for 90 min before flow cytometric analysis. Potential extracellular FCS-born Separase-unrelated proteolytic activity was inhibited by a cocktail of peptidase inhibitors (PImix) as measured by cell lysate-based assay. Separase proteolytic activity was monitored as released Rh110 fluorescence. Corresponding Western blot immunostaining experiments (below) illustrate the expression levels of main Separase regulatory proteins (i.e. CyclinB1, Securin) that reversely correlate with Separase activity. Actin served as loading control. Cell cycle profiles were analyzed by flow cytometry after propidium iodide staining. The percentage of cells in G2/M as a measure of mitotic progression is depicted by the square dotted line. All assays were performed at least in triplicates. (C) Comparative analysis of DNA content in Rh110-negative (upper panel) and positive (lower panel) U937 cells after Hoechst 33342 staining simultaneously performed within the Separase assay. Abbreviations: FCS, fetal calf serum; fluo/cell, fluorescence per cell; 1n, haploid cells; 2n, diploid cells.
Fig 4.
Comparative cleavage characteristics of the peptidic substrates ([Cy5-D-R-E-I-M-R]2-Rh110 and ([Cy5-D-R-E-I-M-D]2-Rh110 in U937 cells.
The Separase-specific conjugated peptidic substrate (DREIMR) is shown in green, the control substrate lacking the Separase cleavage consensus (DREIMD) is depicted in red. (A) Substrate cleavage in the cell extract-based Separase assay as monitored by released Rh110 fluorescence. (B) FACS measurement of Rh110 positive cells after incubation with substrates according to the standardized protocol (90 min at 37°C). (C) Calculation of mean proteolytic activity per cell as measured by released Rh110 fluorescence in the FACS assay. (D) Changes (Δ-values) of proteolytic activities on substrates DREIMR and DREIMD after 48 h of Espl1 silencing when compared to mock treated U937 cells. Corresponding Western blot immunostaining experiments (below) illustrate the knockdown of the Separase protein levels (siRNA (-), 100%; siRNA (+), 24%). Actin served as loading control. Abbreviations: DREIMD, ([Cy5-D-R-E-I-M-D]2-Rh110; DREIMR, ([Cy5-D-R-E-I-M-R]2-Rh110.
Fig 5.
Efflux and influx dynamics of unconjugated Rh110.
A mixed pool of U937 cells representing about 95% negative and 5% Rh110 positive cells (staining: 10 μM for 90 min) was incubated for 90 min while successively monitoring Rh110 fluorescence at given time points (0, 1, 3, 5, 10, 20, 30, 60, and 90 min). The time-related Rh110 fluxes from stained to unstained cells are visualized by colored histogram overlay. Abbreviations: RFU, relative fluorescence units; min, minutes.
Fig 6.
Influence of unspecific extracellular proteases on Separase assay results.
Rh110 fluorescence in U937 cells (A) and in PBMC of a patient with chronic myeloid leukemia (B) as well as in the corresponding cell supernatants were assayed by flow cytometry and lysate-based assay, respectively. Three protease inhibitors alone (Pefabloc SC, soybean trypsin-chymotrypsin inhibitor, MMP-2/MMP-9 inhibitor III) or in combination (PImix) were used to suppress extracellular Separase-unrelated proteolytic activities according to the recommendations of the manufacturers. Proteolytic activity was measured as released Rh110 fluorescence. Abbreviations: RPMI, RPMI-1640 cell culture medium; PI, protease inhibitor; MMP9, matrix metalloproteinase 9, Pefa, Pefabloc SC; Tryp/Chym, trypsin-chymotrypsin inhibitor; PImix, cocktail of all combined peptidase inhibitors. All assays were performed at least in triplicates.
Fig 7.
Comparability of flow cytometric- (A) and lysate-based (B) Separase activity assays.
Cell lines with high (MEG01) and low (BV173) levels of Separase activity and U937 cells at distinct time points (90, 180, 270 min) after release from the nocodazole block were comparatively analyzed in parallel. For (A) the mean Rh110 fluorescence was calculated for all Separase positive cells. Data derived from the lysate-based assay (B) were calculated with respect to levels of Actin that served as internal standard (lysate loading control). Abbreviations: RFU, relative fluorescence units.
Fig 8.
Visualization of Separase proteolytic activity on single cell level in ficollized PBMCs of healthy donors (n = 3) and CML patients (n = 3).
A flow cytometry event-derived dot blot (A) represents Separase active cells ordered by their Rh110 fluorescence. The distribution of Rh110 intensities has been accentuated by coloring cells above the 99.5 percentile (= 0.5% of Separase positive cells) in red and cells below the 99.5 percentile (= 95.5% of Separase positive cells) in blue. The quotient of mean Rh110 fluorescence intensities (mean0.5%/mean99.5%) was calculated to serve as numerical value of cellular Separase activity distribution in samples under investigation (B). Abbreviations: RFU, relative fluorescence units.