Name: detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans
Uploaded: 2018-07-30T13:00:00
Description: Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

The C. elegans strains used in this study were obtained from the Caenorhabditis Genetics Center, which is supported by the National Institutes of Health - Office of Research Infrastructure Programs (P40 OD010440). This work was supported by NIH: 1R03AG048578-01 to Jun Liang, CUNY-CCRG 1501 to Jun Liang, and PSC-CUNY 66184-00 44 and 67248-00 45 to Jun Liang. We thank Cathy Savage-Dunn for kindly sharing her laboratory space. All fluorescence images were collected at Queens College Core Facility. We thank William J. Rice for his comments on the manuscript.

1. Solutions
<ol>
	<li>NGM plates
	<ol>
		<li>In a 2 L Erlenmeyer flask, add 3 g of NaCl, 17 g of agar, 2.5 g of Peptone, and 975 mL of dH2O. Cover the mouth of the flask with aluminum foil. Autoclave the flask for 50 min. Cool it for 20&#8211;30 min.</li>
		<li>Then add sterilized solutions: 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in ethanol, 1 mL of 1 M MgSO4, and 25 mL of 1 M KPO4 (pH 6.0) buffer. Swirl the solutions to mix them well.</li>
		<li>Usi...

<ol>
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This article presented a fast and efficient method to verify protein subcellular changes from the cytoplasm to the nucleus. The protein expression was shown by a GFP fusion, while the nucleus structure was verified by DAPI staining (Figure 3). Since immunostaining C. elegans proteins is challenging, most C. elegans protein subcellular localizations are characterized by tagging them with marker proteins such as GFP, Laz, mCherry, and others18,</s...

A change of protein subcellular localization is a common mechanism in response to internal or external signals such as heat stress, starvation, oxidative stress, apoptosis, protein phosphorylation, and others. For example, heat stress induces a FOXO member DAF-16 nuclear translocation1,2, and the pro-apoptotic BCL-2 protein BID translocates to the mitochondria upon receiving death signaling3,4. Various techniques are available to detect these changes. A combination of western blotting and biochemically isolating subcellular structures (e.g., mitochondria or the nuclei) could well achieve the goal3. However, it requires a specific antibody against the protein of interest. Thus, a well-established antibody becomes the key to the success. An alternative approach is to label different subcellular structures or organelles with various markers such as green fluorescence protein (GFP), red fluorescence protein (RFP), yellow fluorescence protein (YFP), and mCherry, and meanwhile label the protein of interest with other markers. Then, observe them under a confocal microscope to localize the targets5,6. Radioactive isotopes are an alternative choice for labeling target proteins and then detecting their subcellular localization7. However, this method requires proper training and handling of radioactive wastes. Under circumstances such as the lack of a specific antibody, the absence of a proper marker, or the scarceness of equipment such as a confocal microscope, an alternative approach needs to be considered. To identify a protein nuclear translocation, it is attractive to only label the target proteins with a marker and to stain nuclei with the chemical reagent 4',6-diamidino-2-phenylindole (DAPI) since this only requires a regular fluorescence microscope. 
Immunolabeling C. elegans with antibodies is challenging due to the low permeability of either the eggshell or the collagenous cuticle surrounding the animal. Meanwhile, since C. elegans proteins are significantly divergent from their vertebrate orthologs, a few commercial companies provide C. elegans with specific products. It is difficult for a small laboratory to generate C. elegans antibodies for themselves. Researchers in the community often use tagged proteins as markers to demonstrate a protein localization or gene expression. This article uses EXL-1::GFP as an example to track a protein nuclear translocation under heat stress8. An integrated translational exl-1::gfp into the animal genome is used to stably express the gene with gfp fusion. Research showed that exl-1 is expressed in intestine, body wall muscle, and other subcellular structures8. This protocol demonstrates how to synchronize worms to the fourth larval (L4) stage, perform a heat stress experiment, and conduct DAPI staining, ethanol fixation, acetone fixation, and imaging under a regular fluorescence microscope.

<table>
	<tbody>
		<tr>
			<td>DAPI</td>
			<td>Biotium</td>
			<td>40043</td>
			<td>both Hoechst 33258 and Hoechst 33342 also works well</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>CGC</td>
			<td>OP50</td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Kim Wipe</td>
			<td>Kimberly-Clark Corporation</td>
			<td></td>
			<td>soft tissue</td>
		</tr>
		<tr>
			<td>Zeiss&#160; AX10</td>
			<td>Zeiss</td>
			<td></td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiovision Rel 4.8</td>
			<td>Zeiss</td>
			<td></td>
			<td>microscope software</td>
		</tr>
		<tr>
			<td>AxioCam MR Rev3</td>
			<td>Zeiss</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-20 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Chloride intracellular channel proteins (CLIC) are multifunctional proteins that are highly conserved across species12. Much research shows that CLICs regulate cellular stress, autophagy, apoptosis, carcinogenesis, angiogenesis, and the macrophage innate immune response in the mammalian system13,14,15,16,17</s...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

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.

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 ...

This method can help answer key questions in the C.elegans field, such as cell biology. The main advantage of this technique is that it is fast and efficient to obtain signals of nuclear staining, GFP, and sub-cellular localization changes simultaneously. To begin, generate an extrachromosomal array align with a translational construct of target genes.Alternatively, obtain such lines from the Caenorhabditis Genetic Center, which is a public resource for C.elegans research. Or, from a previously a previously published research laboratory. After exposing worms to gamma radiation to integrate the extrachromosomal lines into the genome, select the stably expressed lines so that each animal expresses a marker protein such as a GFP or roller.To remove mutations generated during the radiation, outcross the lines at least two times. Use the resulting transgenic line to detect protein expression pattern changes under stress. Next, after preparing NGM worm plates according to the text protocol, streak an OP50 clone and culture the bacteria in liquid LB broth overnight.Seed the NGM plates with liquid OP50 culture and incubate the plates in a 37 degree Celsius incubator overnight to grow bacteria lawn as a food source for the worms. Cool down the plates at room temperature for at least 30 minutes before use. Grow the transgenic line at 20 degrees Celsius without starvation, for at least two generations prior to the heat stress assay.Pick eight to ten young, gravid adults to a new worm plate. Then let them lay eggs for about three to five hours, and remove all the adults from the plates. To synchronize the worms, let the eggs hatch and grow the larvae for approximately 48 hours to the fourth larval, or L4 stage.That can be identified by the vulva, which is a half-moon structure. Then heat-stress the worms by incubating the plates with the L4 larvae at 35 degrees Celsius for two to five hours. Include a thermometer in an incubator to accurately measure the temperature.Pipette 10 microliters of M9 onto the center of a glass slide. Then pick heat-shocked worms and transfer them into the drop of buffer. Alternatively, use M9 to wash the worms off the plate.Then centrifuge the sample at 1000 times gravity at room temperature for one minute. Discard the supernatant, then add the worms to the glass slide. Under a dissecting microscope, use a soft tissue to drain any excess liquid.Then, while still under the microscope, add 10 microliters of 95%alcohol onto the worms and continue to observe them. Immediately after the ethanol dries, repeat the addition of ethanol two more times. It is critical towards the process of the ethanol evaporating from the animals under the dissecting microscope.Once the ethanol comes off the animals, immediately proceed to the next step. Add 10 microliters of DAPI to the worms. Immediately apply a cover slip and seal the slide with transparent nail polish gel.After allowing the nail polish to set for approximately 10 minutes, view the animals under a fluorescence microscope. To fix animals with acetone, use M9 to wash off the heat-shocked plate and centrifuge the sample at 1000 times gravity at room temperature for one minute. Discard the supernatant and use one milliliter of distilled water to wash the pellet.After removing the supernatant, add 400 microliters of 30%acetone and incubate the sample for 15 minutes. Then centrifuge the tube at 1000 times gravity at room temperature for one minute. After discarding the supernatant, use 500 microliters of distilled water to wash the animals two times.Add 200 microliters of DAPI to the pellet, or four times the amount of the total worms'volume to the tube, and incubate the sample for 15 minutes. Centrifuge the sample and discard the supernatant. Then use 500 microliters of distilled water to wash the pellet two times before mounting and imaging as before.To carry out imaging, turn on the UV light source and the fluorescence microscope. Then turn on the computer connected to the microscope. Load the slide onto the fluorescence microscope.Use a 10X objective lens to locate the worms. Then increase the magnification power to 400X and focus on the specimen. Next, open the software provided with the microscope.Click on Acquisition, then click on Camera, and select the camera connected to the microscope. Under the same Acquisition, click on Multidimensional Acquisition. This opens a new Multidimensional Acquisition navigation window.Click the multi channel button and all available channels will appear. Choose blue, green, and DIC to observe the specimen. In the Multidimensional Acquisition navigation window, leave the blue DAPI, green GFP, and gray DIC channels on, and right-click on other channels, such as red Rhodamine and white Brightfield, to close them.To measure the exposure time for each channel, left-click the blue DAPI channel to select it and click measure"to take a live image with an automated exposure time. On the left side of the live image window, manually adjust the exposure time if desired, then click OK.Set up the exposure times for the GFP and DIC channels in a similar manner. In the Multidimensional Acquisition navigation window, click on Start"to automatically collect three images under the three different channels, in order, with the set exposure times.To save the file, in the main menu, click on File, Save As.Input the file name and the folder. Save the file as the default file format to preserve the detailed imaging information for future reference. To export the file in other formats, under File, click Export.Set the file name and folder. Check Create a project folder"to better organize the data. After choosing a desired file type from the drop-down menu, click Start"to export.There are two chloride intracellular channel protein homologues in C.elegans, exl-1 and exc-4. A recent study showed that exl-1 regulates animal stress management. Here, a translational exl-1 GFP construct was integrated into an animal genome, and transgenic lines were created, which stably express green fluorescent exl-1.Under heat stress, exl-1 GFP accumulates in the nucleus in the intestinal region as a strong GFP signal overlaps with the nucleus. To confirm the subcellular localization of exl-1, DAPI staining was carried out in both ethanol and acetone fixed animals. Both methods show clear nuclei in the animal body and the overlapping GFP and DAPI signals confirm that exl-1 GFP indeed accumulated in the nuclei in the intestinal region.Once mastered, this technique can be done in one hour if it is performed properly. After watching this video, you should have a good understanding of how to perform a quick fixation to preserve the GFP signal and protein nuclear translocation under stress in C.elegans.

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...