HaloTag technology for specific and covalent labeling of fusion proteins.

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging.

BACKGROUND: Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity. NEW METHOD: In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long-term. RESULTS: We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration. COMPARISON WITH EXISTING METHODS: Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact. CONCLUSIONS: These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.

HaloTag as a reporter gene: positron emission tomography imaging with (64)Cu-labeled second generation HaloTag ligands.

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.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology.

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

A Rho GTPase signal treadmill backs a contractile array.

VIDEO ABSTRACT: Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying "signal treadmill" in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2-powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays, and proper functioning of these gradients is dependent on both contraction and F-actin turnover.

HaloTag: a novel reporter gene for positron emission tomography.

Among the many molecular imaging techniques, reporter gene imaging has been a dynamic area of research. The HaloTag protein is a modified haloalkane dehalogenase which was designed to covalently bind to synthetic ligands (i.e. the HaloTag ligands [HTL]). Covalent bond formation between the HaloTag protein and the chloroal-kane within the HTL occurs rapidly under physiological conditions, which is highly specific and essentially irreversible. Over the years, HaloTag technology has been investigated for various applications such as in vitro/in vivo imaging, protein purification/trafficking, high-throughput assays, among others. The goal of this study is to explore the use of the HaloTag protein as a novel reporter gene for positron emission tomography (PET) imaging. By attaching a HaloTag -reactive chloroalkane to 1, 4, 7-triazacyclononane-N, N', N"-triacetic acid (NOTA) through hydrophilic linkers, the resulting NOTA-conjugated HTLs were labeled with (64)Cu and tested for PET imaging in living mice bearing 4T1-HaloTag-ECS tumors, which stably express the HaloTag protein on the cell surface. Significantly higher uptake of (64)Cu-NOTA-HTL-S (which contains a short hydrophilic linker) in the 4T1-HaloTag-ECS than the non-HaloTag-expressing 4T1 tumors was observed, which demonstrated the HaloTag specificity of (64)Cu-NOTA-HTL-S and warranted future investigation of the HaloTag protein as a PET reporter gene.

Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation.

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.

A microtubule-dependent zone of active RhoA during cleavage plane specification.

Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

Concentric zones of active RhoA and Cdc42 around single cell wounds.

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.