The specific and rapid labeling of cell surface proteins with recombinant FKBP-fused fluorescent proteins

The presence and abundance of proteins in the plasma membrane (PM) is regulated by exocytosis and endocytosis. Exocytosis is required for the maintenance of cellular homeostasis following stimulation, whereas endocytosis is important for the reuse and degradation of PM and receptor proteins. Through the specific labeling of proteins on the PM, the process of endocytosis can be tracked and monitored in living cells. Several genetic tags and site-specific labeling approaches that involve the coupling of small organic molecules to tag-fused proteins through either self-labeling or enzymatic ligation have been developed for this purpose However, these methods require a long time for an efficient labeling. Techniques with slow labeling rates are not suitable for live cell imaging. This is especially true for tracking proteins that are rapidly cycled to and from the PM in neurons because the integrity and function of these proteins require extremely fast membrane trafficking. A typical neuron in the human brain fires action potentials at 10 Hz, causing the fusion of several hundred synaptic vesicles every second (Sudhof, 2004). Accordingly, the rapid endocytosis of extensive transmem-brane proteins following their exocytosis is required for the local regeneration of synaptic vesicles at the synaptic terminals. At present, fast endocytosis cannot be effectively traced using the available chemical probe-based labeling methods. Sun et al. have recently reported a fast-labeling variant of the SNAP-tag, termed SNAP f , which displays up to a tenfold increase in its reactivity towards benzylguanine substrates (Sun et al., 2011). The time required for 50% labeling of SNAP f (t 1/2) is 11 to 34 s for different SNAP-surface BG dyes (Sun et al., 2011). However, the widespread use of SNAP f remains limited by the background fluorescence from unre-acted or non-specifically bound substrates. Moreover, a thorough wash step is required to reduce the fluorescence signals of unreacted dyes for the vast majority of chemical labeling processes, including SNAP f-tag labeling; this requirement restricts certain labeling applications, such as the real-time imaging of the endocytosis of membrane proteins. To overcome this difficulty, Sun et al. developed fluo-rogenic SNAP-tag probes with low background fluorescence that become highly fluorescent only upon reaction with their target proteins (Sun et al., 2011). However, the incorporation of intramolecular quenchers greatly reduces the reactivity of the fluorogenic substrates for both SNAP-tag and SNAP f-tag (Sun et al., 2011), resulting in a slow labeling rate, with t 1/2 ranging from 3 to 18 …

Application FKBP-AP21967 to VAMP2 endocytosis. (a) Basal VAMP2 representing constitutive insulin secretion was first labeled with mKate2-FKBP-AP21967. Stimulated VAMP2 representing regulated insulin secretion was labeled with GFP-FKBP-AP21967. They colocalized very well after entering the cells, indicating both VAMP2 endocytosis shared the same pathway. (b) PM basal VAMP2 endocytosis underwent clathrin-dependent pathway as transferrin. (c) VAMP2 was internalized and trafficking simultaneously with transferrin in the same vesicle as indicated by the arrows.  Movie S1 mKate2-FKBP-AP21967 labeling VAMP2-FRB and transferrin-488 endocytosis in the same vesicles in the live cells.

General Methods
The rapalog AP21967 and cDNA for FKBP and FRB (T2098L) was a kindly gift from ARIAD pharmaceuticals, and ascomycin was obtained from Sigma-Aldrich. Pfu polymerase, restriction enzymes, T4 DNA ligase and SNAP-Surface 488 were obtained from NEB. The primers for PCR and Alexa Fluor 488-conjugated transferrin were purchased from Invitrogen. Unless otherwise noted, all of the remaining chemical reagents were purchased from Sigma.
The concentrations of the recombinant proteins were measured on a Biodropsis UV spectrophotometer (Wuzhou Beijing Orient). The microscopy experiments were performed on an FV1000 confocal laser scanning microscope (Olympus Optical Co., Tokyo, Japan). The experiments were conducted using a 60× oil objective. The images were processed using the FV1000 software package FluoView and Image J (NIH).

The cloning, expression and purification of pRSETa-mKate2-FKBP
The plasmid pRSETa-mKate2-FKBP was constructed as follows: The fluorophore mKate2 was amplified by PCR with primers mKate2-up (AAA GGA TCC ATG GTG AGC GAG CTG ATT AAG) and mKate2-rev (CTG CAG GGT GCC GCC AGA GCC TCC TCT GTG CCC CAG TTT GCT AGG). FKBP was amplified with the primers FKBP-up (GGA GGC TCT GGC GGC ACC CTG CAG GGA GTG CAG GTG GAA ACC ATC TC) and FKBP-rev (AAA GAA TTC TTA ATG CAT TTC CAG TTT TAG AAG CTC CAC). The mKate2 and FKBP PCR products were mixed, and the primers mKate2-up and FKBP-rev were used to perform PCR to generate the mKate2-FKBP chimera. The mKate2-FKBP fragment was digested with BamH1 and EcoR1 and inserted into pRSETa. This plasmid was transformed into E. coli TOP10 competent cells and grown overnight on LB agar plates with ampicillin. Colonies from the agar plates were transferred into 5 ml LB with ampicillin, grown overnight at 37°C and then sequenced (Invitrogen) to confirm that the construct was correct.
The cells were grown on agar plates containing ampicillin, picked the single clone and transferred into 5 ml LB with ampicillin, and grown overnight at 37°C. Subsequently, 500 mL of LB medium was inoculated with the 5 ml overnight cultures and grown at 37 °C at 200 rpm until the optical density (OD) at 600 nm reached 0.6 ~ 0.7. IPTG (1 mM) was added for protein induction, and the culture was further grown at 20 °C for 8 h. The cells were collected by centrifugation at 8000 g for 5 min. The pellet was then resuspended in 30 mL of lysis buffer (50 mM Tris, 500 mM NaCl and 5 mM imidazole, pH 8.0) and lysed by sonication on ice. The mixture was centrifuged at 18,000 g for 30 min, yielding a clear lysate containing the target protein mKate2-FKBP. The lysate was loaded in a Ni-NTA column (Qiagen) that was previously balanced three times with lysis buffer. The loaded column was then washed three times with wash buffer (50 mM Tris, 500 mM NaCl and 50 mM imidazole, pH 8.0) to remove the nonspecific binding proteins. Elution buffer (50 mM Tris, 500 mM NaCl and 500 mM imidazole, pH 8.0) was added to the column to elute mKate2-FKBP, and the flow-through was collected.
The fraction containing mKate2-FKBP was further purified in PBS by gel filtration chromatography using a Superdex 200 column (GE Healthcare). The purified mKate2-FKBP protein was then concentrated in a centrifugal concentrator with a molecular weight cut-off of 10 kDa (Millipore) and stored at -80°C. The concentration of mKate2-FKBP was determined by measuring the absorbance at 280 nm with a Biodropsis UV spectrophotometer (Wuzhou Beijing Oriental).

VAMP2 endocytosis
INS-1 cells transfected with VAMP2-TagBFP-FRB (T2098L) were manipulated as described for VAMP2 translocation. After the cells were labeled with EGFP-FKBP for 30 min and washed three times with PBS, they were incubated in PBS for another 15 min to allow the endocytosis of the two fractions of the VAMP2 molecules. The cells were subjected to FV1000 fluorescence microscopy with 405 nm, 488 nm and 559 nm lasers used for image acquisition.
INS-1 cells were transfected with VAMP2-TagBFP-FRB (T2098L). The transfected cells were starved for 2 hours in serum-free RPMI-1640 in the presence of 1 mg/ml BSA at 37°C. The cells were first incubated in PBS containing 1 μM mKate2-FKBP mixed with 1 μM AP21967 for 1 min to label the VAMP2 molecules.
The cells were then washed three times with PBS and then incubated with 10 μg/ml Alexa Fluor® 488-conjugated transferrin (Invitrogen) for 20 min at 37°Cmin. The cells were then subjected to fluorescence microscopy after washes with PBS three times.
Hela cells expressing VAMP2-TagBFP-FRB (T2098L) were starved for 2 hours in serum-free DMEM in the presence of 2 mg/ml BSA at 37°C. The cells with the highly expression level of TagBFP-FRB were selected and subjected to confocal microscope spin-disk. The cells were first incubated in PBS containing 1 μM mKate2-FKBP mixed with 1 μM AP21967 for 1 min to label the VAMP2 molecules.
The cells were then washed three times with PBS and incubated with 30 μg/ml Alexa Fluor® 488 Conjugate transferrin (Invitrogen). The images were acquired in real time with lasers at 488 nm and 561 nm on a Zeiss Observer Z1 microscope (Carl Zeiss) with a 60 × oil objective. The exposure time is 200 ms for 488 nm and 300 ms for 561 nm.
The gain of EM-CCD was set at 200.