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 CagedGl...

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, th...

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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

Research

JoVE Journal

Chemistry

10.4K Views.  University of Tokyo. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van de levering van geneesmiddelen, zoals hoe een geneesmiddel te leveren aan een specifieke cellulaire-korrelig. Het belangrijkste voordeel van deze techniek is dat nucleaire translocatie van gas kan worden veroorzaakt door licht. Het aantonen van de procedure zal Rina Mogaki en Akio Arisaka de grad studenten uit mijn groep.Om te beginnen met de voorbereiding van de quantum dot gekoppelde kooi moleculair...

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 ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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

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

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.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

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

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