The β1Int-HaloTag fusion protein was well tolerated by multiple cell types, including mammalian cell lines and human neural stem cells [22
]. Immunocytochemistry with β1 integrin and HaloTag antibodies showed that the β1Int-HaloTag fusion protein was expressed at the cell membrane in a similar pattern to endogenous β1 integrin (Figure ). Fixed cells were non-permeabilized to show that the HaloTag reporter protein was localized on the cell surface.
To study membrane proteins in live cells with the HaloTag technology, we developed a fluorescent ligand which should not cross the cell membrane. To make this ligand cell impermeable, we added a negatively charged dye to the standard activated linker. To confirm that this novel ligand, HaloTag®
488 (HaloTag 488), was cell impermeable, we labeled cells expressing HaloTag on the surface or only inside. Live cell imaging showed that the HaloTag 488 ligand specifically labeled the cell-surface HaloTag protein in cells stably expressing β1Int-HaloTag, but did not label the intracellular protein in cells stably expressing HaloTag fused to a nuclear localization sequence [23
] (Figure ). This confirms that the novel ligand is cell impermeable and that the surface HaloTag protein fused to integrin can functionally bind ligands.
To reveal the β1Int-HaloTag protein topology and subcellular distribution, we used HaloTag ligands with a modified fluorescence protease protection (FPP) assay [24
]. The FPP assay, described by Lorenz (2006), determines the topology and localization of proteins in living cells by monitoring trypsin-induced destruction of GFP attached to a protein of interest. We separately labeled surface and internal protein pools of β1Int-HaloTag in live cells with the cell impermeable fluorescent ligand, HaloTag 488, followed by the cell permeable fluorescent ligand, HaloTag TMR (Figure ). Spatial separation of protein pools is depicted by a green rim around a red interior (Figure ). Trypsin exposure to live cells stripped the external HaloTag 488 ligand over time, but preserved the internal HaloTag TMR ligand (Figure ). This result shows that the HaloTag protein fused to integrin was orientated on the surface of the cell membrane, and that the multi-functional HaloTag technology can be used to determine topology of membrane proteins. In some instances, β1Int-HaloTag labeled on the surface with the HaloTag 488 ligand was internalized before trypsin addition. Unlike the surface exposed protein removed by trypsin, this recycled protein showed fluorescence protease protection (Additional Figure 1
Figure 3 Revealing protein topology and subcellular localization. (a) Diagram of separately labeled surface and internal protein pools of β1Int-HaloTag, and trypsin exposure to strip the surface labeled pool. (b) Pre trypsin treatment, live HEK293 cells (more ...)
To reveal the β1Int-HaloTag protein subcellular localization, we combined the HaloTag technology and the permeabilization agent digitonin [24
]. Cells were co-transfected with β1Int-HaloTag and GFP, and then labeled with the HaloTag TMR ligand (Figure ). Co-expression of the proteins is shown by the yellow overlay (Figure ). Digitonin treatment permeabilized the membrane, which allowed freely floating GFP to diffuse out of the cell over time while internally bound β1Int-HaloTag was retained in the cell, presumably by cellular transport and recycling machinery (Figure ). This result shows that the HaloTag fusion did not affect normal processing of a membrane bound protein, and that the HaloTag technology can be used to distinguish free floating and membrane bound proteins.
Membrane proteins, such as integrins, are typically trafficked through secretory and endocytic pathways [25
]. Permeabilization experiments showed β1Int-HaloTag was retained in the cell, but to confirm that the β1Int-HaloTag fusion protein was associated with intracellular transport and recycling organelles, we combined fluorescent HaloTag ligands to label live cells with fixed cell immunocytochemistry. To assess protein delivery to the membrane, live cells expressing β1Int-HaloTag were labeled with the HaloTag TMR ligand, and then processed for immunocytochemistry to visualize the endoplasmic reticulum (ER), ER-intermediate golgi complex (ERIGC), or golgi (Figure ). To assess protein recycling from the membrane, live cells expressing β1Int-HaloTag were labeled with the HaloTag 488 ligand, and then processed for immunocytochemistry to visualize the early and late endosomes (Figure ). Co-localization of the β1Int-HaloTag protein with cellular transport machinery is shown by the yellow overlay, and suggests that fusing HaloTag to the truncated integrin does not alter normal protein flow through secretory and endocytic pathways.
Figure 4 Trafficking and internalization of membrane proteins. (a) HeLa cells transiently expressing β1Int-HaloTag were labeled with the HaloTag TMR ligand, then fixed and processed for immunocytochemistry to visualize the endoplasmic reticulum (ER), ER-golgi (more ...)
We used the HaloTag technology and immunocytochemistry to also show that the β1Int-HaloTag protein co-localized with expected membrane proteins, such as cadherin and transferrin receptors [20
]. To assess protein co-localization at the membrane, live cells expressing β1Int-HaloTag were labeled with the HaloTag 488 ligand, and then processed for immunocytochemistry (Figure ). Co-localization of the β1Int-HaloTag protein with cadherin is shown by the yellow overlay, and suggests that β1Int-HaloTag fusion not only traffics properly to the membrane but also co-localizes with other expected membrane proteins. To assesses protein co-internalization from the membrane, live cells expressing β1Int-HaloTag were labeled with the HaloTag 488 ligand and a transferrin Alexa Fluor®
594 conjugate (Figure ). Co-localization of the β1Int-HaloTag protein with the transferrin receptor is shown by the yellow overlay, and suggests that the β1Int-HaloTag fusion not only internalizes through the proper cellular machinery, but also co-internalizes with expected membrane proteins.
Mature integrins at the cell membrane are glycosylated, and the internal pool is partially glycosylated as it traffics through the secretory pathway [30
]. To confirm that the β1Int-HaloTag protein not only traffics normally through the secretory pathway but also undergoes proper post-translational modification, we used fluorescent HaloTag ligands followed by SDS-PAGE analysis. Live cells expressing β1Int-HaloTag were sequentially labeled with HaloTag 488 and TMR ligands to separately label surface and internal proteins, respectively (Figure ). Lysate from labeled cells showed two distinct protein pools on SDS-PAGE in lane 2 (Figure ). This band pattern could be because the higher molecular weight green surface pool of β1Int-HaloTag is heavily glycosylated compared to the red intracellular pool. To confirm whether the distinct protein pools were due to differential protein glycosylation, we glycanase treated cells lysates. Deglycosylation by either O- or N-glycanase treatment caused a significant shift in the green surface pool of β1Int-HaloTag (lane 3 and 4). Conversely, O-glycanase treatment caused no visible shift in the red internal pool of β1Int-HaloTag (lane 3), and N-glycanase treatment caused only a minor shift (lane 4). Glycanase treatment of the HaloTag reporter protein produced no band shift on SDS-PAGE (data not shown), suggesting that the shift in β1Int-HaloTag is due to glycosylation of the integrin protein. Our results show that the surface pool of β1Int-HaloTag is glycosylated, which is expected for integrins and suggests that fusing HaloTag to a truncated integrin does not alter proper post-translational glycosylation.
Figure 5 Spatial and temporal separation of proteins using HaloTag Technology. (a) HEK293 cells stably expressing β1Int-HaloTag were sequentially labeled with the HaloTag 488 and TMR ligands and then rinsed and lysed. Lysate was non-glycanase treated (lane (more ...)
We also used the HaloTag technology to follow protein modification over time. Cells expressing β1Int-HaloTag were labeled sequentially with HaloTag 488 and TMR ligands, and lysate was then collected immediately or up to 12 hours after labeling (Figure ). As expected, lysate from cells labeled with the HaloTag TMR ligand alone showed two protein pools (lane 1) and lysate from cells labeled with the HaloTag 488 ligand alone showed only the higher molecular weight protein pool (lane 2). Substantiating figure , lysate from cells sequentially labeled with both ligands showed separation of the two protein pools at early time points (lanes 3–5). However, over time the lower band for the red internal pool shifted up, presumably as this protein arrived at the membrane in a glycosylated form (lanes 6–9) [31
]. Additionally, the upper band for the green surface pool disappeared, presumably as this protein was endocytosed and degraded. The overlaid SDS-PAGE shows that the HaloTag technology can be used to track different protein pools and monitor post-translational modifications over time.
Finally, we used the HaloTag technology and live cell imaging to visualize spatial separation and real-time translocation of β1Int-HaloTag. Cells expressing β1Int-HaloTag were labeled sequentially with HaloTag 488 and TMR ligands. Live cell imaging showed two distinct protein pools, with the surface protein labeled specifically with the HaloTag 488 ligand and the intracellular protein labeled with the HaloTag TMR ligand (Figure ). Re-imaging 12 hours after labeling shows that the initial red cytoplasmic pool has moved to the membrane and the initial green surface pool has internalized (Figure ). Timelapse imaging shows real-time translocation after labeling, which begins with the green surface pool internalizing at 1 hour, continues with the red internal pool trafficking to the surface, and ends with the red surface pool internalizing (Additional Figure 2
). The HaloTag technology can show clear translocation of two separate protein pools in live cells, and these results of β1Int-HaloTag movement corroborate the immunocytochemistry and SDS-PAGE results.
With the HaloTag technology, functional reporters such as fluorescent ligands can be used to image live cells, and affinity handles such as biotin can be used to capture cells expressing HaloTag on the surface. We co-expressed β1Int-HaloTag, or HaloTag as a control, with luciferase. Live cells were labeled with HaloTag PEG-Biotin ligand and then captured on streptavidin coated plates. Luciferase assay results show the specific capture of β1Int-HaloTag-expressing cells compared to HaloTag-expressing control cells (Figure ). Specific capture of β1Int-HaloTag-expressing cells was blocked when streptavidin coated plates were pre-coated with HaloTag PEG-Biotin. In addition to the in vitro luciferase assay, live cell imaging confirms that β1Int-HaloTag-expressing cells can be specifically captured using the HaloTag technology and that captured cells survive. Live cells were labeled with HaloTag PEG-Biotin ligand, captured on streptavidin coated plates, and then replated for live cell imaging. Labeling plated cells with the HaloTag TMR ligand shows the survival of specifically captured β1Int-HaloTag-expressing cells compared to control cells (Figure ).
Figure 6 Cell capture using HaoTag Technolgy. (a) HeLa cells co-expressing β1Int-HaloTag and luciferase were labeled with HaloTag PEG-Biotin. They show specific capture on SA plates compared to labeled cells co-expressing HaloTag and luciferase. Pre-coating (more ...)