Name: spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags
Uploaded: 2019-01-17T13:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

We acknowledge the Center for NanoBio Integration, the University of Tokyo. This work was supported by Grant-in-Aid for Young Scientists (B) (26810046) to K.O. and partially supported by Grant-in-Aid for Specially Promoted Research (25000005) to T.A. R.M. thanks the Research Fellowships of Japan Society for the Promotion of Science (JSPS) for Young Scientists and the Program for Leading Graduate Schools (GPLLI).

1. Preparation of Guests with CagedGlue-R Tags
<ol>
	<li>Prepare CagedGlue-NBD solution.
	<ol>
		<li>Synthesize CagedGlue-NBD (Figure 1) following the procedures previously described20.</li>
		<li>Prepare a stock solution of CagedGlue-NBD (10 mM) in dry dimethyl sulfoxide (DMSO). 
		Note: Store the stock solution in dark. The solution can be diluted with aqueous buffers or cell culture media upon usage.</li>
	</ol>
	</li>
	<li>Prepare CagedGlue-QD solution.
	<ol>
		<li>Synthesize CagedGlue-dibenzocylooctyne (CagedGlue-DBCO) (Figure 1) following the procedures previously described20.</li>
		<li>Prepare a stock solution of CagedGlue-DBCO (10 mM) in dry DMSO.</li>
		<li>For the preparation of CagedGlue-QD, first prepare azide-functionalized QDs (Azide-QD; Figure 3). Add 100 &#181;L of a dimethyl formamide (DMF, 125 &#181;M) solution of azide-PEG4-NHS ester (Figure 3) to 400 &#181;L of a DMF (500 nM) solution of quantum dots (QDs) coated with amine-functionalized PEG (Amine-QD; Figure 3). Stir the mixture for 1 h at room temperature.</li>
		<li>Dialyze the resulting solution for 24 h against 800 mL of DMF using a regenerated cellulose membrane with 3,500 molecular weight cut-off (MWCO).</li>
		<li>Dilute the stock solution of CagedGlue-DBCO to 50 &#181;M with DMF. Add 200 &#181;L of the solution to the post-dialysis solution (Figure 3) and stir the mixture for 3 h at room temperature.</li>
		<li>Dialyze the resulting solution for 24 h against 800 mL of DMF using a regenerated cellulose membrane (25,000 MWCO).</li>
		<li>Dilute the resulting solution to 200 nM with DMF.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58631/58631fig3.jpg" /> 
Figure 3: Schematic illustration of the preparation of CagedGlue-QD. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of Hep3B Cell Sample for Microscopic Observations
<ol>
	<li>Maintain human hepatocellular carcinoma Hep3B cells in Eagle&#8217;s minimal essential medium (EMEM) containing 10% fetal bovine serum (FBS) at 37 &#176;C under 5% CO2.</li>
	<li>Seed the cells the day before the experiment. Seed 5.0 &#215; 103 Hep3B cells per well of an 8-chambered glass substrate in EMEM (10% FBS, 200 &#181;L), and incubate the cell sample at 37 &#176;C under 5% CO2 for 24 h.</li>
	<li>Remove the culture medium and rinse the cell sample with 100 &#181;L of Dulbecco&#8217;s phosphate buffer saline (D-PBS) twice.</li>
</ol>
3. Observation of Nuclear Translocation of Small-molecule Guests Triggered by UV Light
<ol>
	<li>Supply the cell sample (prepared in step 2.3) with 200 &#181;L of FBS-free EMEM containing CagedGlue-NBD (10 &#181;M) and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 3 h. 
	Note: Incubation of the cell sample in FBS-free EMEM for longer than 4 h causes serious cell damage.</li>
	<li>Remove the culture medium and rinse the cell sample with 100 &#181;L of D-PBS twice.</li>
	<li>For visualization of the endosomes, supply the cell sample with 200 &#181;L of EMEM (10% FBS) containing a red-fluorescent dye (e.g., LysoTracker Red, 100 nM), and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 20 min. Remove the culture medium, and rinse the cell sample with 100 &#181;L of D-PBS twice. Supply the cell sample with 200 &#181;L of EMEM (10% FBS).</li>
	<li>For nuclear translocation of CagedGlue-NBD, expose the cell sample to UV light for 2 min via an optical fiber using a 100-W xenon light source equipped with a 365 nm bandpass filter. For a reference cell sample without UV exposure, keep the cell sample in dark. 
	Note: The lid of the glass substrates can be taken off for efficient UV exposure. Long-time exposure to UV light may cause cytotoxicity to the cells.</li>
	<li>For visualization of the nuclei, add 1 &#181;L of Hoechst 33342 (1 mg/mL) to the culture medium, and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 10 min.</li>
	<li>Subject the cell sample to confocal laser scanning microscopy and record the micrographs upon excitation at 488 nm (&#955;obs = 500-530 nm), 543 nm (&#955;obs = 565-620 nm), and 710 nm (two-photon; &#955;obs = 390-465 nm) for NBD, red-fluorescent dye, and Hoechst 33342, respectively.</li>
</ol>
4. Observation of Nuclear Translocation of Small-molecule Guests Triggered by Two-photon NIR Light
<ol>
	<li>Supply the cell sample (prepared in step 2.3) with 200 &#181;L of FBS-free EMEM containing CagedGlue-NBD (10 &#181;M) and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 3 h.</li>
	<li>Remove the culture medium and rinse the cell sample with 100 &#181;L of D-PBS twice. Supply the cell sample with 200 &#181;L of EMEM (10% FBS).</li>
	<li>Subject the cell sample to confocal laser scanning microscopy and record the micrographs upon excitation at 488 nm (&#955;obs = 500-530 nm).</li>
	<li>For nuclear translocation of CagedGlue-NBD, irradiate the region including the cell of interest with a two-photon excitation laser (710 nm), installed as a light source in the microscope, for 2 min (30 s &#215; 4). Observe the translocation as described in step 4.3.</li>
</ol>
5. Observation of Nuclear Translocation of Macromolecular Guests Triggered by UV Light
<ol>
	<li>Supply the cell sample (prepared in step 2.3) with 200 &#181;L of FBS-free EMEM containing CagedGlue-QD (10 nM), and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 3 h.</li>
	<li>Remove the culture medium and rinse the cell sample with 100 &#181;L of D-PBS twice. Supply the cell sample with 200 &#181;L of EMEM (10% FBS).</li>
	<li>For nuclear translocation of CagedGlue-QD, expose the cell sample to UV light for 2 min via an optical fiber using a 100-W xenon light source equipped with a 365 nm bandpass filter. For a reference cell sample without UV exposure, keep the cell sample in dark.</li>
	<li>For visualization of the nuclei, add 1 &#181;L of Hoechst 33342 (1 mg/mL) to the culture medium, and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 10 min.</li>
	<li>Subject the cell sample to confocal laser scanning microscopy and record the micrographs upon excitation at 405 nm (&#955;obs = 430-520 nm) and 488 nm (&#955;obs = 625-680 nm) for Hoechst 33342 and QDs, respectively.</li>
</ol>
6. Cell Viability Assay
<ol>
	<li>Maintain human hepatocellular carcinoma Hep3B cells in EMEM (10% FBS) at 37 &#176;C under 5% CO2.</li>
	<li>Seed the cells the day before the experiment. Seed 5.0 &#215; 103 Hep3B cells per well of a 96-well culture plate in EMEM (10% FBS, 200 &#181;L), and incubate the cell sample at 37 &#176;C under 5% CO2 for 24 h.</li>
	<li>Remove the culture medium and rinse the cell sample with 100 &#181;L of D-PBS twice.</li>
	<li>Supply the cell sample with 200 &#181;L of FBS-free EMEM containing CagedGlue-NBD (0.1-100 &#181;M) and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 3 h.</li>
	<li>Expose the cell sample to UV light for 2 min via an optical fiber using a 100-W xenon light source equipped with a 365 nm bandpass filter. For an analogous cell sample without UV exposure, keep the cell sample in dark.</li>
	<li>Add 10 &#181;L of Cell Counting Kit-8 reagent (10 &#181;L) to the culture medium and incubate the resulting cell sample at 37 &#176;C under 5% CO2 for 2 h.</li>
	<li>Subject the cell sample to absorption spectroscopy (&#955; = 450 nm) using a microplate reader.</li>
</ol>

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The authors have nothing to disclose.

Previous investigations of light-triggered translocation of proteins into the cell nucleus have been achieved using caged NLS7,8,9. As mentioned earlier, these methods require additional techniques to incorporate the NLS-tagged proteins into the cytoplasm. In contrast, our CagedGlue-R tag enables not only photo-induced nuclear translocation but also cellular uptake of the guests. This feature of the CagedGlue-R tag is advantageous for in vivo applications.
The CagedGlue-R tag can deliver macromolecular guests such as QDs into the cell nucleus. The QDs employed here are larger in diameter (DH = 15-20 nm) than the nuclear pores (~5 nm)24, suggesting the possibility to deliver other macromolecules that cannot passively diffuse into the nucleus. Protocols for functionalization of biomacromolecular surfaces with azide groups are well established25; in addition, synthesis of CagedGlue-R tags bearing other functional groups such as maleimides, N-hydroxysuccinimide (NHS) esters, and so forth as an anchoring unit will broaden the applicability of CagedGlue-R tags to various biomacromolecules.
Needless to say, the critical step of this method is the incorporation of the guests tagged with CagedGlue-R via endocytosis. The efficiency of the cellular uptake of the tagged guests depends on their concentration and the incubation time. If guests of interest, when tagged with CagedGlue-R, are endocytosed inefficiently, incubate the cells longer with higher concentration of the tagged guests. A possible alternative is to increase the number of CagedGlue-R incorporated into the guest.
The guests used in this protocol are covalently conjugated to the CagedGlue-R tag. This is a potential disadvantage especially for delivery of small-molecule ligands, since their binding to the target biomolecules may be inhibited by the bulky dendritic tag. Incorporation of a stimuli-responsive linker26 between the CagedGlue-R tag and the guest molecule may allow release of the guests after nuclear translocation.
In summary, we demonstrated light-triggered nuclear translocation of guests using photoactivatable caged molecular glue (CagedGlue-R) tags. The guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in the endosomes. Upon photoirradiation, CagedGlue-R is converted to UncagedGlue-R, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. Further long-time observations may be important to investigate the fate of the guests that remain in the endosomes as well as those delivered into the cell nucleus. Spatiotemporally controlled gene expression using CagedGlue-R tags is an interesting subject worthy of further investigation.

The cell nucleus, which carries genetic information, is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases including cancer and genetic disorders1,2,3. For nuclear delivery of drugs, conjugation of peptide tags such as nuclear localization signals (NLS)4,5,6 has been widely investigated. However, in order to reduce undesired side effects, spatiotemporal control of the nuclear translocation is necessary.
Previously, light-triggered translocation of proteins into the cell nucleus has been achieved using caged NLS7,8,9. NLS migrates into the cell nucleus by binding to cytoplasmic transport proteins6. In the reported methods, guest proteins bearing caged NLS are directly incorporated into the cytoplasm by microinjection8 or expressed in the target cells using a genetic code expansion technique9. Therefore, a method that can achieve both cellular uptake and photo-induced nuclear translocation is advantageous for practical applications.
Herein, we describe light-triggered nuclear translocation of guests in living cells using dendritic caged molecular glue (CagedGlue-R, Figure 1) tags. Water-soluble molecular glues10,11,12,13,14,15,16,17,18,19,20,21,22,23 bearing multiple Gu+ pendants have been previously developed, which tightly adhere to proteins11,12,13,14,15,16,17, nucleic acids18,19,20, phospholipid membranes21, and clay nanosheets22,23 through the formation of multiple salt bridges between their Gu+ pendants and oxyanionic groups on the targets. The Gu+ pendants of CagedGlue-R are protected by an anionic photocleavable group, butyrate-substituted nitroveratryloxycarbonyl (BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and stay in endosomes (Figure 2). Upon photoirradiation, the BANVOC groups of CagedGlue-R are detached to yield an uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which then facilitates the migration of the tagged guest into the cytoplasm followed by the cell nucleus (Figure 2). The CagedGlue-R tag can be uncaged by exposure to UV or two-photon near-infrared (NIR) light without serious phototoxicity. We demonstrate the spatiotemporally controlled nuclear delivery of macromolecular guests as well as small-molecule guests with CagedGlue-R tags, using quantum dots (QDs) and a fluorescent dye (nitrobenzoxadiazole; NBD), respectively, as examples.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58631/58631fig1.jpg" /> 
Figure 1: Schematic structures of CagedGlue-R. The 9 guanidinium ion (Gu+) pendants of CagedGlue-R are protected by a butyrate-substituted nitroveratryloxycarbonyl (BANVOC) group. The BANVOC groups are cleaved by irradiation with UV or two-photon NIR light. The focal core of CagedGlue-R is functionalized with either nitrobenzoxadiazole (NBD) or dibenzocylooctyne (DBCO). Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58631/58631fig2.jpg" /> 
Figure 2: Schematic illustration of light-triggered nuclear translocation of guests conjugated with a CagedGlue-R tag. The guest/CagedGlue-R conjugate is taken up into living cells via endocytosis. Upon photoirradiation, the CagedGlue-R tag is uncaged to yield an UncagedGlue-R tag, which can facilitate endosomal escape of the tagged guest. Subsequently, the tagged guest migrates into the cell nucleus. Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Azide-PEG4-NHS ester</td><td>Click Chemistry Tools</td><td>AZ103</td><td></td></tr><tr><td>Q-dot 655 ITK</td><td>Invitrogen</td><td>Q21521MP</td><td></td></tr><tr><td>Regenerated cellulose membrane (MWCO 3,500)</td><td>NIPPON Genetics</td><td>TOR-3K</td><td></td></tr><tr><td>Regenerated cellulose membrane (MWCO 25,000)</td><td>Harvard Apparatus</td><td>7425-RC25K</td><td></td></tr><tr><td>Hep3B Cells</td><td>ATCC</td><td>HB-8064</td><td></td></tr><tr><td>8-chambered glass substrate</td><td>Nunc</td><td>155411JP</td><td></td></tr><tr><td>96-well culture plate</td><td>Nunc</td><td>167008</td><td></td></tr><tr><td>Eagle&#039;s minimal essential medium (EMEM)</td><td>Thermo Fisher Scientific</td><td>10370-021</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GE Healthcare</td><td>SH30406.02</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffer saline (D-PBS)</td><td>Wako Pure Chemical Industries</td><td>045-29795</td><td></td></tr><tr><td>LysoTracker Red</td><td>Lonza Walkersville</td><td>PA-3015</td><td></td></tr><tr><td>Hoechst 33342</td><td>Dojindo</td><td>H342</td><td></td></tr><tr><td>Cell Counting Kit-8</td><td>Dojindo</td><td>CK04</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl-Zeiss</td><td>LSM 510</td><td>Equipped with two-photon excitation laser (Mai Tai laser, Spectra-Physics)</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>TCS SP8</td><td></td></tr><tr><td>Xenon light source</td><td>Asahi Spectra</td><td>LAX-102</td><td></td></tr><tr><td>Microplate reader</td><td>Molecular Devices</td><td>SpectraMax Paradigm</td><td></td></tr></tbody></table>

spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.

Before photoirradiation, Hep3B cells incubated with CagedGlue-NBD exhibited punctate fluorescence emission from their interior (&#955;ext = 488 nm; Figures 4A and 4C, green). An analogous micrograph was obtained upon excitation at 543 nm for red-fluorescent dye (Figures&#160;4B and 4C, red), indicating that CagedGlue-NBD localized in the endosomes. Accordingly, the fluorescence emission assignable to CagedGlue-NBD (Figure 4D, green) was observed outside the cell nucleus, which was visualized with Hoechst 33342 (&#955;ext = 710 nm, two-photon; Figure 4D, blue). After UV irradiation, the cells emitted fluorescence due to NBD (Figure 4E, green) also from the nucleus (Figure 4E, blue), suggesting that CagedGlue-NBD was uncaged to yield UncagedGlue-NBD, which migrated into the cytoplasm followed by the cell nucleus. Such nuclear translocation of CagedGlue-NBD can be induced site-selectively by two-photon NIR light (Figure 5A and Movie 1, white dashed circle). As shown in Figure 5B and Movie 1, CagedGlue-NBD in nonirradiated areas did not escape from the endosomes and remained as punctate fluorescence. No appreciable cytotoxicity was observed for the cells treated with CagedGlue-NBD before and even after the UV exposure (Figure 6).
The CagedGlue-R tags can also deliver macromolecular guests such as QDs into the cell nucleus. The QD/CagedGlue-R conjugate (CagedGlue-QD, Figure 3) can be taken up into Hep3B cells (Figure 7A). Visualization of the cell nucleus with Hoechst 33342 indicates that CagedGlue-QD remains outside of the nucleus before photoirradiation (Figure 7A). After UV exposure for 2 min, the fluorescence emission of QDs emerged in the nucleus (Figures 7B and 7C). A cross-sectional image of the cells demonstrated that the fluorescence emission assignable to QDs is indeed emitted from inside the nucleus (Figure 7D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58631/58631fig4.jpg" /> 
Figure 4: Endosomal escape and nuclear translocation of CagedGlue-NBD triggered by UV light. Confocal laser scanning micrographs of Hep3B cells after 3-h incubation at 37 &#176;C in EMEM containing CagedGlue-NBD (10 &#181;M) followed by rinsing with D-PBS. (A, B) Micrographs were recorded upon excitation at (A) 488 nm (&#955;obs = 500-530 nm, green) and (B) 543 nm (&#955;obs = 565-620 nm, red) after 20 min incubation in EMEM (10% FBS) containing red-fluorescent dye (100 nM). (C) Merged image of (A) and (B). (D, E) Micrographs recorded upon excitation at 488 nm (&#955;obs = 500-530 nm, green) and 710 nm (two-photon; &#955;obs = 390-465 nm, blue). The Hep3B cells, treated with CagedGlue-NBD, were incubated at 37 &#176;C in EMEM (10% FBS) containing Hoechst 33342 (5 &#181;g/mL) before (D) and after (E) 2-min UV exposure at 365 nm. Scale bars = 20 &#181;m. Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58631/58631fig5.jpg" /> 
Figure 5: Nuclear translocation of CagedGlue-NBD triggered by two-photon NIR light. Confocal laser scanning micrographs of Hep3B cells after 3-h incubation at 37 &#176;C in EMEM containing CagedGlue-NBD (10 &#181;M) followed by rinsing with D-PBS. Micrographs were recorded upon excitation at 488 nm (&#955;obs = 500-530 nm) before (A) and after (B) two-photon irradiation at 710 nm for 2 min (30 s &#215; 4). The white dashed circle in (A) represents the irradiated area. Scale bars = 20 &#181;m. Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58631/58631fig6.jpg" /> 
Figure 6: Cell viability assay. Viabilities of Hep3B cells after incubation in EMEM containing CagedGlue-NBD (0.1-100 &#181;M) before (A) and after (B) 2-min UV exposure at 365 nm. Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58631/58631fig7.jpg" /> 
Figure 7: Nuclear translocation of CagedGlue-QD triggered by UV light. Confocal laser scanning micrographs of Hep3B cells upon excitation at 405 nm (&#955;obs = 430-520 nm) and 488 nm (&#955;obs = 625-680 nm). Hep3B cells were incubated at 37 &#176;C for 3 h in EMEM containing CagedGlue-QD (10 nM), rinsed with D-PBS, and then incubated at 37 &#176;C for 10 min in EMEM (10% FBS) containing Hoechst 33342 (5 &#181;g/mL) before (A) and after (B, C) 2-min exposure to UV light at 365 nm. (D) Cross-sectional image along the yellow line in (C). Scale bars = 20 &#181;m. Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie" src="/files/ftp_upload/58631/58631movie1.jpg" /> 
Movie 1. Nuclear translocation of CagedGlue-NBD triggered by two-photon NIR light. Time-lapse confocal laser scanning microscopy of Hep3B cells after 3-h incubation at 37 &#176;C in EMEM containing CagedGlue-NBD (10 &#181;M) followed by rinsing with D-PBS. Micrographs were recorded with 3-s intervals upon excitation at 488 nm (&#955;obs = 500-530 nm). The cells located in the white dashed circle were irradiated with two-photon NIR light at 710 nm for 2 min (30 s &#215; 4). Reprinted with permission from reference20. <a href="https://www.jove.com/files/ftp_upload/58631/Movie1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is ...

spatiotemporally-controlled-nuclear-translocation-guests-living-cells

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.3K Views.  University of Tokyo. This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.

Video: Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengineering

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Biology

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Immunology and Infection

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.