Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter

Promega Corporation 2800 Woods Hollow Road, Madison, WI 53711, USA.
BMC Cell Biology (Impact Factor: 2.34). 02/2008; 9(1):17. DOI: 10.1186/1471-2121-9-17
Source: PubMed


The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag, to the extracellular domain of a truncated integrin.
Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture.
The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.

Download full-text


Available from: Georgyi Los,
  • Source
    • "By selecting suitable ligands the same tag can be used for live cell microscopy, immunofluorescence, Western Blotting, protein purification and co-precipitation assays [16], [17], [19]–[26]. Moreover, the Halotag allows following the accumulation and/or the degradation of the protein of interest by two-color pulse/chase experiments with high temporal resolution [27]. Lastly, the Halotag has the advantage of not possessing glycosylation sites, that could affect folding and transport of the chimeric proteins in the secretory compartment. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.
    PLoS ONE 10/2014; 9(10):e108496. DOI:10.1371/journal.pone.0108496 · 3.23 Impact Factor
  • Source
    • "As a versatile tool, the HaloTag technology has attracted much attention for a broad array of biomedical applications such as in vitro optical imaging [1] [2] [3], in vivo cell labeling/imaging [4] [5], protein purification/trafficking [6] [7], study of protein-protein and protein-DNA interactions [8], analysis of protein stability [9], highthroughput assays [10], single molecule force spectroscopy [11], ribosome tagging [12], among many others [13]. The HaloTag technology involves two key components: the HaloTag protein and HaloTag ligands (HTLs). "
    [Show abstract] [Hide abstract]
    ABSTRACT: THE GOAL OF THIS STUDY IS TO EMPLOY THE HALOTAG TECHNOLOGY FOR POSITRON EMISSION TOMOGRAPHY (PET), WHICH INVOLVES TWO COMPONENTS: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N'N''-triacetic acid). Microscopy studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-HTL2G-S/L. Uptake of (64)Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1, and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) at early time points. In comparison, (64)Cu-NOTA-HTL2G-S did not demonstrate significant uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates confirmed that (64)Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors, corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with (64)Cu-NOTA-HTL2G-L serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET, as well as many other studies of gene/protein/cell function in vivo.
    American Journal of Translational Research 05/2013; 5(3):291-302. · 3.40 Impact Factor
  • Source
    • "By combining a cell permeable ligand in one color with a non-permeable ligand of different color we can differentially label surface exposed proteins where HaloTag is exposed on the cellular surface, from the intracellular pool of the same protein. This affords analysis of protein trafficking, endocytosis and study of membrane protein biology [32]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding protein function and interaction is central to the elucidation of biological processes. Systematic analysis of protein interactions have shown that the eukaryotic proteome is highly interconnected and that biological function frequently depends on the orchestrated action of many proteins. Perturbation of these functions or interactions can lead to various disease states and pharmacologic intervention can result in corrective therapies. The fact that proteins rarely act in isolation, but rather comprise complex machines that stably and/or transiently interact with many different partners at different times, demands the need for robust tools that allow comprehensive global analyses of these events. Here we describe a powerful protein fusion technology, the HaloTag platform, and how it enables the study of many facets of protein biology by offering a broad choice of applications. We review the development of the key aspects of the technology and it's performance in both in vitro and in vivo applications. In particular, we focus on HaloTag's multifunctional utility in protein imaging, protein isolation and display, and in the study of protein complexes and interactions. We demonstrate it's potential to help elucidate important facets of proteomic biology across complex biological systems at the biochemical, cell-based and whole animal level.
    Current Chemical Genomics 12/2012; 6(1):72-8. DOI:10.2174/1875397301206010072
Show more