Engineering and production of Rab5 activity fluorescent biosensor
Rab5 presented on the inner leaflet of the plasma membrane, early and intermediate endosomes or macropinosome is important for both clatherin-dependent, receptor-mediated endocytosis and clatherin-independent, fluid-phase uptake. Rab5 GTPase is activated by guanine nucleotide exchange factors (GEFs) and deactivated by GTPase activating proteins (GAPs). The activation of Rab5 GTPase accelerates the rates of nucleotide exchange in Rab5 GTP binding site and the Rab5-bound GTP hydrolysis (15
). The translocation of Rab5 GTPase between cytosol and inner membranes is facilitated by GDP dissociation inhibitor (GDI) and GDI displacement factors (GDFs). Targeting of Rab5 GTPases to specific organelles also depends on GEFs, effectors and GAPs (16
) (, panel a).
Figure 1 Design and production of Rab5 activity fluorescent biosensor (RAFB). a) schematic Rab5 GTPase cycles between inactive (GDP-bound) and active (GTP-bound) states. b) scheme of RAFB peptide precursor. c, scheme of the preparation of the QD-conjugated RAFB. (more ...)
We chose the Rab5 binding domain (R5BD) of rabaptin 5 (Rab5 effector) as the active Rab5 recognition moiety of the Rab5 activity fluorescent biosensor (RAFB). The cDNA of the 124 amino acid peptide sequence (17
) (, panel b) was subcloned into the GST-fusion protein expression vector pGEX-4T-2, resulting in a GST-R5BD fusion protein construct with the GST sequence at the 5′ end (, panel b). The six amino acids thrombin cleavage sequence (LVPRGS) is derived from the vector, and the 9 amino acids polyarginine sequence (RRRRRRRRR) was introduced at the N-terminus of the R5BD by PCR to increase the RAFB cytoplasmic membrane entering efficiency (18
). An 11 amino acids (PCHPQFPRCYA) biotin mimetic sequence (BMS) (19
) was genetically fused to the carboxyl terminus of the R5BD for subsequent fluorophore conjugation through binding to streptavidin. Quantum dot (QD), a nanocrystal widely utilized in fluorescence imaging due to its superior fluorescent properties, was chosen to prepare the first probe (20
). The 45 KD GST-R5BD fusion protein was pulled down in the cell lysate by the GSH-coated Sepharose 4B beads (Supplementary Figure 1
). The GST-R5BD-BMS fusion protein was directly conjugated with streptavidin-coated QD525. The final probe was generated by thrombin cleavage and the QD-conjugated RAFB was recovered from the supernatant and quantified by the BCA assay (, panel c).
Quantification of active Rab5 in brain tissues using QD-conjugated RFAB
We first tested the RAFB-QD525 for imaging Rab5 activity on the tissues of a Ts65Dn mouse, the most widely used mouse model for Down syndrome (DS) (21
). Elevated expression of amyloid precursor protein has been linked to neurodegenerative processes in Ts65Dn mice (22
). Additionally, abnormally high levels of Rab5 activity have been observed in Ts65Dn mouse tissues possibly due to the mis-regulated endocytotic pathway (23
50 μm-thick cryostat cut sections of the 9 month-old DS mouse brains along with the wild-type (2N) controls were permeablized using 0.3% Triton X-100, and immunostained using RAFB-QD525. Polyclonal antibody against Rab5 was used to label the total Rab5 in mouse brain cells, and the fluorescence intensities generated by the immunostaining were used as the index to evaluate the relative amount of total Rab5 in the brain tissues of 2N and DS mice (, panel a-d). The ratio of the average immunostaining fluorescence intensity of over 200 neuron cells between DS and 2N mice in cortex is 1.217 ± 0.024 (, panel e), and 0.882 ± 0.018 in hippocampus (, panel g), indicating the difference in the total Rab5 between DS and 2N neurons in both cortex and hippocampus is relatively small (p<0.0001) and the difference between the cortex and hippocampus is not statistically significant (P=0.6533). However, the level of active Rab5 (Rab5-GTP) in DS mouse brain is much higher than the 2N mice, as revealed by the RAFB-QD525 probe. Imaging analysis of single cortical and hippocampal neurons rendered the same observation: the elevated Rab5 activity in the DS mouse model, but similar total Rab5 level in both genotypes (, panel c and d). The active Rab5 levels represented by fluorescence intensity from RAFB-QD525 in 2N and DS mouse brain samples were similarly quantified by analyzing over 200 RAFB-stained cells. We found that in the DS mouse model (i.e. Ts65Dn), the average level of GTP-bound active Rab5 was increased by 4.5-fold in the cortices (, panel f) and by 1.8-fold in the hippocampi (, panel h), compared with the 2N mice. We have further generated the 3D simulated graphic images to depict the different Rab5 active level in cortices and hippocampi for both DS and 2N mice (, panel i).
Figure 2 Measurement and quantification of Rab5 activity in disease mouse model by RAFB. Cortical and hippocampal slices of DS mice and littermates (2N) were immunostained simultaneously with polyclonal Rab5 antibody (Rab5) and RAFB-QD525 (Rab5-GTP). a, b) fluorescence (more ...)
In order to examine the general tissue structure within the cortical and hippocampal areas, we used 0.5% thionin to stain the paraffin-embedded tissue sections through the cortical and hippocampal regions of the brain in Ts65Dn and 2N mice. Thionin stains DNA and Nissl substance, which is primarily ribosomal RNA. The staining is widely used as an indicator of general status of brain cells. No apparent abnormalities were found in both cortical and hippocampal regions for DS and 2N mouse brains, respectively, from the thionin staining (, panel j). We have also shown before that Ts65Dn mice brain is devoid of any plaques and tangles, and no increased levels of amyloid has been found in these mice (22
). Together with the RAFB imaging data, these results suggest that the abnormal active Rab5 level may occur earlier before the appearance of other pathophysiological hallmarks of AD.
Pre-quenched RAFB measures Rab5 activity in live cells and real time
Rab5 has been reported to regulate endocytosis and set the pace of membrane fission and fusion between plasma membrane and inner membranes (24
). Abnormally high Rab5 activity perturbs the homeostasis between membrane fission and fusion, thus compromises vesicle trafficking and endosome-associated signal transduction, creating pathogenic conditions in the cell (26
). We next developed the RAFB to image active Rab5 in single live cells in real time.
To apply the RAFB for live-cell imaging, we have designed a pre-quenching module to keep the RAFB from emission during the endocytosis process after internalization The pre-quenched RAFB will regain fluorescence emission after it leaves the endosome and enters the cytoplasm (27
). To have RAFB pre-quenched during the membrane docking and endocytosis steps before it reaches the cytoplasm and binds the active GTP-bound Rab5 at the cytosolic surface of the endosome (, panel a) will presumably minimize the imaging background, because it is often difficult to wash out excessive amount of RAFB under the live-cell imaging conditions once RAFB gets internalized inside the cell. Furthermore, many pathophysiological conditions lead to compromised endocytotic pathways which prevent endosome sorting and maturation, hence stalling the endosomal cargo contents inside the abnormal endocytotic compartments instead of releasing them into the cytosol (28
). In this case the Rab5 activity imaging could be inaccurate due to possible false positive fluorescence from the aggregated inner membranes, and the stuck RAFB within the aggregates.
Figure 3 Design, synthesis and verification of the pre-quenched fluorophore moiety of cytoplasm-activatable RAFB. a) the pre-quenched RAFB is internalized into early endosome and then released into the cytoplasm where it gets activated and binds active Rab5. b) (more ...)
To keep the RAFB quenched before reaching the cytoplasm from endosomes, we introduced a dabcyl quencher moiety in the sensor. We chose fluorescein isothiocyanate (FITC) as the fluorophore in order to maximize the dabcyl quenching efficiency meanwhile maintain the optimal fluorescence quantum yield at the neutral pH environment in the cytoplasm where Rab5 is activated. The dabcyl quencher was connected via the disulfide bond through the side chain of cysteine, so once in the cytoplasm the abundant glutathione would reduce the disulfide bond to release the dabcyl quencher (, panel b). The addition of biotin to the FITC-Cys-Dabcyl complex through lysine enables its conjugation to the R5B-BMS fusion peptide through the binding to streptavidin.
The biotinylated Lys(Dde)-Cys(Trt)-OH was synthesized by conventional solid phase peptide synthesis using Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH and biotin as the building blocks subsequently. After the removal of the Dde protecting group, FITC was introduced to the free ε-amine of Lysine. Then the resulting peptide was cleaved from the resin and purified by reverse-phase high performance liquid chromatograph (HPLC) (, panel c). The product was confirmed by the MALDI-TOF mass spectrometer (Supplementary Figure 2
, panel d).
We have first examined the quenching and activation efficiency of the FITC-Dabcyl unit after the synthesis. For in vitro test, series dilution of the Biotin-Lys(FITC)-Cys(S-S-Dabcyl) (from 0.1 μM to 5 μM) were treated with 5 mM of reduced L-glutathione at 37 °C for 5 min to simulate the cytosolic condition. The fluorescence emission spectra (from 510 nm to 650 nm) were collected, and the Biotin-Lys(FITC)-Cys(S-S-Dabcyl) has little emission at all concentrations. Addition of glutathione (GSH) produced a large (up to 2,500 fold) increase in its emission (, panel d). For in vivo test, we measured the fluorescence emission at 520 nm of HeLa cells incubated with different concentrations of Biotin-Lys(FITC)-Cys(S-S-Dabcyl) (0, 0.1, 0.5, 1, 2, 5 μM). DMEM culture medium was used as a negative control, and all samples were incubated at 37 °C for 15 min with 5% CO2 before measurement. Strong fluorescence emission was detected in HeLa cell samples (F-D/cell, orange columns, , panel e) in comparison to the samples incubated in DMEM culture medium without cells (F-D/medium, green columns, , panel e). The fluorescent intensity increases up to 400 fold after activation in cells. These results suggest that the pre-quenched Biotin-Lys(FITC)-Cys(S-S-Dabcyl) can be efficiently activated by intracellular glutathione.
Biotin-Lys(FITC)-Cys(S-S-Dabcyl) was then conjugated with the recombinant R5BD-BMS peptide to generate the pre-quenched RAFB for intracellular Rab5 activity imaging. The GST-R5BD-BMS fusion protein was pull-down by GSH coated Sepharose 4B beads from expressing E. coli lysate, and streptavidin (SA) was added at 1:1 molar ratio to the beads slurry after 3 times PBS wash. Equivalent moles of Biotin-Lys(FITC)-Cys(S-S-Dabcyl) was then added to bind with SA, and the R5BD-BMS-SA-Biotin-Lys(FITC)-Cys(S-S-Dabcyl) biosensor (pre-quenched RAFB) was cleaved out by thrombin from the beads and recovered in the aqueous phase (, panel a).
Figure 4 Imaging Rab5 activity in live cells with pre-quenched RAFB. a) scheme of preparation steps: expression of recombinant protein, pre-quenched fluorophore moiety conjugation and purification of pre-quenched RAFB. b) imaging active Rab5 in live HeLa cells (more ...)
A control RAFB was generated by mutating the valine 822 into aspartic acid in the R5BD domain of the RAFB using site-directed mutagenesis to produce a V822D isoform (R5BD(V822D)-BMS) that does not bind to GTP-bound active Rab5 (17
). Similar conjugation of this mutant with Biotin-Lys(FITC)-Cys(S-S-Dabcyl) through SA afforded the negative probe RAFB-Mut as a control for live-cell imaging of Rab5 activity.
At the basal level in normal conditions, the active form of Rab5 in vivo
accounts for just a small fraction of total Rab5 (15
). Diseased cells show increased Rab5 activation, presumably because activation and suppression of Rab5 cycle is disturbed by perturbations of the homeostasis of Rab5-GTP binding, hydrolysis, and recycling between endosomal membrane and cytoplasm (30
). We simulated the diseased states in mammalian cell cultures by overexpressing different forms of Rab5 to create a set of high and low levels of active Rab5. Two Rab5 mutants were fused with mCherry (a transfection marker) to make the mCherry-fused, constitutively active Rab5Q79L-mC and dominantly negative Rab5S34N-mC (31
). The Q79L point mutation abolished the GTPase activity of Rab5, and therefore this mutant could not hydrolyze its bound GTP, leading to constitutive activation of Rab5. Contrary to the Q79L mutation, the Rab5S34N construct has its serine 34 replaced by asparagine, which stops the recharging process of GTP back to Rab5 once it is hydrolyzed and keeps Rab5 staying at the GDP-bound inactive state.
HeLa cells were transiently transfected with Rab5Q79L-mC or Rab5S34N-mC, and incubated with 100 nM of pre-quenched RAFB 18 hours after transfection. As an additional control, Rab5Q79L-mC expressing cells were incubated with 100 nM of pre-quenched non-binding RAFB-Mut. Cells were imaged live without washing after 30 min incubation at 37 °C (, panel b). Strong punctuated green fluorescence signal (corresponding to the Rab5 activity) was observed in red fluorescent cells (indicating expression of constitutively active Rab5) when stained with RAFB (, panel b, top row). In contrast, minimal green fluorescent signal was detected in constitutively active Rab5 expressing cells when stained with the negative control RAFB-Mut (, panel b, bottom row). The average fluorescence intensity in cells stained with the RAFB is 65-fold higher than that in cells stained with the RAFB-Mut (, panel c). This result confirms that the point mutation of V822D in the R5BD effectively disabled the binding between the RAFB and the active Rab5 and the observed fluorescence from the pre-quenched RAFB correlates with the level of active Rab5 in cells.
A very modest level of fluorescence signal was detected from the dominantly negative Rab5 expressing cell line when stained with the pre-quenched RAFB (, panel b, middle row). The average fluorescence intensity in Rab5S34N-mC expressing cells is 11-fold lower than that in cells expressing constitutively active Rab5 (Rab5Q79L-mC) (, panel c). The low RAFB signal in Rab5S34N-mC expressing cells is consistent with the fact that no exogenous active Rab5 is expressed and the observed weak, evenly distributed fluorescence is likely due to the basal level of endogenous Rab5 activation.
In Rab5Q79L-mC expressing cells, the strong RAFB signals are punctuated, suggesting that they were with aggregated endosomes with active Rab5 associated on the surface. In Rab5S34N-mC expressing cells, the pre-quenched RAFB reaches the cytosol but cannot associate with endosomes because few active Rab5 at the cytosolic side of endosomes is available to bind. Consequently, the fluorescence of RAFB will be activated by the glutathione reduction in the cytosol, but will be weak, evenly distributed. These results have demonstrated that the pre-quenched RAFB can specifically image active Rab5 in live cells.
In summary, we report here the synthesis and validation of novel Rab5 activity biosensors for the detection of active Rab5 in both fixed tissues and live cells. Taking advantage of the Rab5 binding domain and the pre-quenching strategy, our biosensors are able to image the active form of small G protein Rab5 located on the cytosolic side of the early endosome membrane with minimum background noise (32
). Since both endocytotic and autophagic pathways have been reported to be dysfunctional in different disease models, we expect a wide spectrum of applications for the Rab5 activity-imaging probe. Furthermore, it opens the door to label many signaling pathway regulators during pathogenetic cellular events. An array of downstream effectors of important small G proteins such as Ras, Rho, Rap1, Rac1, RalA, Cdc42, Arf1 and Arf6, could also be analyzed in a similar fashion as with Rab5 by exploiting their corresponding specific effector-binding domains to the active forms of these small G proteins.