Papers - Hanaoka, Kenjiro

Azobenzene-caged sulforhodamine dyes: A novel class of 'turn-on' reactive probes for hypoxic tumor cell imaging

Chevalier A., Piao W., Hanaoka K., Nagano T., Renard P.Y., Romieu A.

Methods and Applications in Fluorescence (Methods and Applications in Fluorescence)  3 ( 4 )  2015.12

Research paper (scientific journal), Accepted

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New sulforhodamine-based fluorescent 'turn-on' probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble 'azobenzene-caged' fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH2) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH2. This valuable fluorogenic response was obtained for the three 'smart' probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH2-Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

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Thiosulfate mediates cytoprotective effects of hydrogen sulfide against neuronal ischemia

Marutani E., Yamada M., Ida T., Tokuda K., Ikeda K., Kai S., Shirozu K., Hayashida K., Kosugi S., Hanaoka K., Kaneki M., Akaike T., Ichinose F.

Journal of the American Heart Association (Journal of the American Heart Association)  4 ( 11 )  2015.11

Research paper (scientific journal), Accepted

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Background-Hydrogen sulfide (H2S) exhibits protective effects in various disease models including cerebral ischemia-reperfusion (I/R) injury. Nonetheless, mechanisms and identity of molecules responsible for neuroprotective effects of H2S remain incompletely defined. In the current study, we observed that thiosulfate, an oxidation product of H2S, mediates protective effects of an H2S donor compound sodium sulfide (Na2S) against neuronal I/R injury. Methods and Results-We observed that thiosulfate in cell culture medium is not only required but also sufficient to mediate cytoprotective effects of Na2S against oxygen glucose deprivation and reoxygenation of human neuroblastoma cell line (SH-SY5Y) and murine primary cortical neurons. Systemic administration of sodium thiosulfate (STS) improved survival and neurological function of mice subjected to global cerebral I/R injury. Beneficial effects of STS, as well as Na2S, were associated with marked increase of thiosulfate, but not H2S, in plasma and brain tissues. These results suggest that thiosulfate is a circulating "carrier" molecule of beneficial effects of H2S. Protective effects of thiosulfate were associated with inhibition of caspase-3 activity by persulfidation at Cys163 in caspase-3. We discovered that an SLC13 family protein, sodium sulfate cotransporter 2 (SLC13A4, NaS-2), facilitates transport of thiosulfate, but not sulfide, across the cell membrane, regulating intracellular concentrations and thus mediating cytoprotective effects of Na2S and STS. Conclusions-The protective effects of H2S are mediated by thiosulfate that is transported across cell membrane by NaS-2 and exerts antiapoptotic effects via persulfidation of caspase-3. Given the established safety track record, thiosulfate may be therapeutic against ischemic brain injury.

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Identification of Tissue-Restricted Bioreaction Suitable for in Vivo Targeting by Fluorescent Substrate Library-Based Enzyme Discovery

Yoshioka K., Komatsu T., Nakada A., Onagi J., Kuriki Y., Kawaguchi M., Terai T., Ueno T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 38 ) 12187 - 12190 2015.09

Research paper (scientific journal), Accepted,  ISSN  00027863

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Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

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Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein and Its Application to Develop a Scaffold for Red Fluorescent Probes

Hirabayashi K., Hanaoka K., Takayanagi T., Toki Y., Egawa T., Kamiya M., Komatsu T., Ueno T., Terai T., Yoshida K., Uchiyama M., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry)  87 ( 17 ) 9061 - 9069 2015.09

Research paper (scientific journal), Accepted,  ISSN  00032700

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Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10′ position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pK<inf>a</inf> inversion, i.e., pK<inf>a1</inf> > pK<inf>a2</inf>. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4′ and 5′ positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase. (Chemical Equation Presented).

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Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport

Terai T., Kohno M., Boncompain G., Sugiyama S., Saito N., Fujikake R., Ueno T., Komatsu T., Hanaoka K., Okabe T., Urano Y., Perez F., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 33 ) 10464 - 10467 2015.08

Research paper (scientific journal), Accepted,  ISSN  00027863

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Artificial ligands of streptavidin (ALiS) with association constants of 106 M-1 were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.

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Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes

Ito H., Terai T., Hanaoka K., Ueno T., Komatsu T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  51 ( 39 ) 8319 - 8322 2015.05

Research paper (scientific journal), Accepted,  ISSN  13597345

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We discovered that positively charged terbium complexes bearing 1,4,7,10-tetraazacyclododecane functionalized with amide ligands are highly sensitive to dynamic luminescence quenching by NAD(P)H. We exploited this phenomenon to establish a general time-resolved luminescence-based assay platform for sensitive detection of NAD(P)H-dependent enzyme activities. This journal is

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Fluorescent probes for hydrogen sulfide (H<inf>2</inf>S) and sulfane sulfur and their applications to biological studies

Shimamoto K., Hanaoka K.

Nitric Oxide - Biology and Chemistry (Nitric Oxide - Biology and Chemistry)  46   72 - 79 2015.04

Research paper (scientific journal), Accepted,  ISSN  10898603

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Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. The most commonly used methods or detecting H2S are the methylene blue method and the electrode method, but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (RSSH) and polysulfides (RSSnSR). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH). Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies.

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Development of a Series of Near-Infrared Dark Quenchers Based on Si-rhodamines and Their Application to Fluorescent Probes

Myochin T., Hanaoka K., Iwaki S., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 14 ) 4759 - 4765 2015.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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Near-infrared (NIR) fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are nonfluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region. They are nonfluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluorescence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes. (Chemical Equation Presented).

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New class of bioluminogenic probe based on bioluminescent enzyme-induced electron transfer: BioLeT

Takakura H., Kojima R., Kamiya M., Kobayashi E., Komatsu T., Ueno T., Terai T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 12 ) 4010 - 4013 2015.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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Bioluminescence imaging (BLI) has advantages for investigating biological phenomena in deep tissues of living animals, but few design strategies are available for functional bioluminescent substrates. We propose a new design strategy (designated as bioluminescent enzyme-induced electron transfer: BioLeT) for luciferin-based bioluminescence probes. Luminescence measurements of a series of aminoluciferin derivatives confirmed that bioluminescence can be controlled by means of BioLeT. Based on this concept, we developed bioluminescence probes for nitric oxide that enabled quantitative and sensitive detection even in vivo. Our design strategy should be applicable to develop a wide range of practically useful bioluminogenic probes.

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Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC)

Sakamoto H., Egashira S., Saito N., Kirisako T., Miller S., Sasaki Y., Matsumoto T., Shimonishi M., Komatsu T., Terai T., Ueno T., Hanaoka K., Kojima H., Okabe T., Wakatsuki S., Iwai K., Nagano T.

ACS Chemical Biology (ACS Chemical Biology)  10 ( 3 ) 675 - 681 2015.03

Research paper (scientific journal), Accepted,  ISSN  15548929

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A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb3+-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

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Development of photo-controllable hydrogen sulfide donor applicable in live cells

Fukushima N., Ieda N., Kawaguchi M., Sasakura K., Nagano T., Hanaoka K., Miyata N., Nakagawa H.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  25 ( 2 ) 175 - 178 2015.01

Research paper (scientific journal), Accepted,  ISSN  0960894X

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Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385 nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

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Synthesis of a photocontrollable hydrogen sulfide donor using ketoprofenate photocages

Fukushima N., Ieda N., Sasakura K., Nagano T., Hanaoka K., Suzuki T., Miyata N., Nakagawa H.

Chemical Communications (Chemical Communications)  50 ( 5 ) 587 - 589 2014.12

Research paper (scientific journal), Accepted,  ISSN  13597345

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We report the design, synthesis and application of a directly photocontrollable hydrogen sulfide (H2S) donor, which releases H2S proportionally to the intensity and duration of photoirradiation. Photocontrolled H2S release from this donor was also demonstrated in bovine serum. This H2S donor should be suitable for use in various biological systems. © 2013 The Royal Society of Chemistry.

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Sodium thiosulfate attenuates acute lung injury in Mice

Sakaguchi M., Marutani E., Shin H.S., Chen W., Hanaoka K., Xian M., Ichinose F.

Anesthesiology (Anesthesiology)  121 ( 6 ) 1248 - 1257 2014.12

Research paper (scientific journal), Accepted,  ISSN  00033022

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Background: Acute lung injury is characterized by neutrophilic inflammation and increased lung permeability. Thiosulfate is a stable metabolite of hydrogen sulfide, a gaseous mediator that exerts antiinflammatory effects. Although sodium thiosulfate (STS) has been used as an antidote, the effect of STS on acute lung injury is unknown. The authors assessed the effects of STS on mice lung and vascular endothelial cells subjected to acute inflammation.

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A design strategy for small molecule-based targeted MRI contrast agents: Their application for detection of atherosclerotic plaques

Iwaki S., Hokamura K., Ogawa M., Takehara Y., Muramatsu Y., Yamane T., Hirabayashi K., Morimoto Y., Hagisawa K., Nakahara K., Mineno T., Terai T., Komatsu T., Ueno T., Tamura K., Adachi Y., Hirata Y., Arita M., Arai H., Umemura K., Nagano T., Hanaoka K.

Organic and Biomolecular Chemistry (Organic and Biomolecular Chemistry)  12 ( 43 ) 8611 - 8618 2014.11

Research paper (scientific journal), Accepted,  ISSN  14770520

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Gadolinium(III) ion (Gd3+) complexes are widely used as contrast agents in magnetic resonance imaging (MRI), and many attempts have been made to couple them to sensor moieties in order to visualize biological phenomena of interest inside the body. However, the low sensitivity of MRI has made it difficult to develop practical MRI contrast agents for in vivo imaging. We hypothesized that practical MRI contrast agents could be designed by targeting a specific biological environment, rather than a specific protein such as a receptor. To test this idea, we designed and synthesized a Gd3+-based MRI contrast agent, 2BDP3Gd, for visualizing atherosclerotic plaques by linking the Gd3+-complex to the lipophilic fluorophore BODIPY to stain lipid-rich environments. We found that 2BDP3Gd was selectively accumulated into lipid droplets of adipocytes at the cellular level. Atherosclerotic plaques in the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits were clearly visualized in T1-weighted MR images after intravenous injection of 2BDP3Gd in vivo.

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Fluorometric assay of integrin activity with a small-molecular probe that senses the binding site microenvironment

Komatsu T., Takeda A., Hanaoka K., Terai T., Ueno T., Tada Y., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  50 ( 100 ) 15894 - 15896 2014.11

Research paper (scientific journal), Accepted,  ISSN  13597345

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We have developed a small-molecular probe, consisting of cyclic RGD pentapeptides bearing a nitrobenzoxadiazole fluorophore at the 4′-residue, which detects integrin αVβ3 activity in terms of fluorescence intensity decrease due to quenching of the fluorophore by interaction with tyrosine-122 at the binding site of the protein. This probe appears to be suitable for a practical, high-throughput fluorescence assay to screen small-molecular modulators of integrin activity. © The Royal Society of Chemistry 2014.

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Practical fluorescence detection of acrolein in human plasma via a two-step tethering approach

Togashi M., Terai T., Kojima H., Hanaoka K., Igarashi K., Hirata Y., Urano Y., Nagano T.

Chemical Communications (Chemical Communications)  50 ( 95 ) 14946 - 14948 2014.11

Research paper (scientific journal), Accepted,  ISSN  13597345

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Acrolein, a cytotoxic α,β-unsaturated aldehyde and disease biomarker, was determined in plasma by means of a novel tethering strategy using Michael addition of the compound to a fluorescent dye, followed by immobilization of the product on microbeads via the aldehyde moiety. Elevation of blood acrolein was detected in mice treated with an anticancer agent cyclophosphamide, which releases acrolein upon activation. This method should be suitable for high-throughput diagnostic and clinical application. © the Partner Organisations 2014.

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Boron dipyrromethene as a fluorescent caging group for single-photon uncaging with long-wavelength visible light

Umeda N., Takahashi H., Kamiya M., Ueno T., Komatsu T., Terai T., Hanaoka K., Nagano T., Urano Y.

ACS Chemical Biology (ACS Chemical Biology)  9 ( 10 ) 2242 - 2246 2014.10

Research paper (scientific journal), Accepted,  ISSN  15548929

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Caged compounds are useful tools for precise spatiotemporal modulation of cell functions, but in most cases uncaging requires ultraviolet (UV) light, which is cytotoxic and has limited tissue penetration. Therefore, caged compounds that can be activated by longer-wavelength light are required. Here we describe a novel photoelimination reaction of 4-aryloxy boron dipyrromethene (BODIPY) derivatives and show that BODIPY can function as a caging group for phenol groups. We developed a novel BODIPY-caged histamine compound, which is photoactivatable with blue-green visible light to stimulate cultured HeLa cells in a spatiotemporally well-controlled manner. This caging strategy is expected to be widely applicable to develop tools for probing various cellular functions. (Chemical Equation Presented).

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Cytoprotective effects of hydrogen sulfide-releasing N-methyl-d-aspartate receptor antagonists mediated by intracellular sulfane sulfur

Marutani E., Sakaguchi M., Chen W., Sasakura K., Liu J., Xian M., Hanaoka K., Nagano T., Ichinose F.

MedChemComm (MedChemComm)  5 ( 10 ) 1577 - 1583 2014.10

Research paper (scientific journal), Accepted,  ISSN  20402503

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Hydrogen sulfide (H2S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection. Cytotoxicity of H2S in neurodegenerative diseases may be mediated by N-methyl-d-aspartate receptor (NMDAR) activation. To exploit cytoprotective effects of H2S while minimizing its toxicity, we synthesized a series of H2S-releasing NMDAR antagonists and examined their effects against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death, a cellular model of Parkinson's disease. We observed that cytoprotective effects of H2S-releasing NMDAR antagonists correlated with their ability to increase intracellular sulfane sulfur, but not H2S, levels. These studies suggest that H2S-donor compounds that increase intracellular sulfane sulfur levels are potentially useful neuroprotective agents against neurodegenerative diseases.

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Thermal or mechanical stimuli-induced photoluminescence color change of a molecular assembly composed of an amphiphilic anthracene derivative in water

Sagara Y., Komatsu T., Terai T., Ueno T., Hanaoka K., Kato T., Nagano T.

Chemistry - A European Journal (Chemistry - A European Journal)  20 ( 33 ) 10397 - 10403 2014.08

Research paper (scientific journal), Accepted,  ISSN  09476539

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Molecular assemblies that change photoluminescence color in response to thermal or mechanical stimulation without dissociation into the monomeric states in water are described herein. A dumbbell-shaped amphiphilic compound forms micellar molecular assemblies in water and exhibits yellow photoluminescence derived from excimer formation of the luminescent core, which contains a 2,6-diethynylanthracene moiety. Annealing of the aqueous solution induces a photoluminescence color change from yellow to green (λ em, max=558→525nm). The same photoluminescence color change is also achieved by rubbing the yellow-photoluminescence-emitting molecular assemblies adsorbed on glass substrates with cotton wool in water. The observed green photoluminescence is ascribed to micelles that are distinct from the yellow-photoluminescence-emitting micelles, on the basis of transmission electron microscopy observations, atomic force microscopy observations, and dynamic light scattering measurements. We examined the relationship between the structure of the molecular assemblies and the photophysical properties of the anthracene derivative in water before and after thermal or mechanical stimulation and concluded that thermal or mechanical stimuli-induced slight changes of the molecular-assembled structures in the micelles result in the change in the photoluminescence color from yellow to green in water. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Rapidly rendering cells phagocytic through a cell surface display technique and concurrent Rac activation

Onuma H., Komatsu T., Arita M., Hanaoka K., Ueno T., Terai T., Nagano T., Inoue T.

Science Signaling (Science Signaling)   ( 334 )  2014.07

Research paper (scientific journal), Accepted,  ISSN  19450877

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Cell surfaces represent a platform through which extracellular signals that determine diverse cellular processes, including migration, division, adhesion, and phagocytosis, are transduced. Techniques to rapidly reconfigure the surface properties of living cells should thus offer the ability to harness these cellular functions. Although the molecular mechanism of phagocytosis is well characterized, the minimal molecular players that are sufficient to activate this elaborate process remain elusive.We developed and implemented a technique to present a molecule of interest at the cell surface in an inducible manner on a time scale of minutes. We simultaneously induced the cell surface display of the C2 domain of milk fat globule epidermal growth factor factor 8 (MFG-E8) and activated the intracellular small guanosine triphosphatase Rac, which stimulates actin polymerization at the cell periphery. The C2 domain binds to phosphatidylserine, a lipid exposed on the surface of apoptotic cells. By integrating the stimulation of these two processes, we converted HeLa cells into a phagocytic cell line that bound to and engulfed apoptotic human Jurkat cells. Inducing either the cell surface display of the C2 domain or activating Rac alone was not sufficient to stimulate phagocytosis, which suggests that attachment to the target cell and actin reorganization together constitute the minimal molecular events that are needed to induce phagocytosis. This cell surface display technique might be useful as part of a targeted, cell-based therapy in which unwanted cells with characteristic surface molecules could be rapidly consumed by engineered cells.

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