Name: studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions
Uploaded: 2016-04-21T13:00:00
Description: Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

The presented work was supported in parts by funding from the Canadian Institutes of Health Research / Fragile X Research Foundation of Canada partnership program to RD, the Jerome Lejeune Foundation to RD, the Interdisziplin&#228;res Zentrum f&#252;r klinische Forschung of the University Erlangen-Nuremberg to RD and from the Deutsche Forschungsgemeinschaft to RD and RE. The authors wish to thank Ingrid Zenger for technical assistance with cell culture maintenance as well as Prof. M. Wegner for making a pCMV5-FLAG vector available. The authors further wish to particularly thank Nadja Schroeder for the helpful support at the video shooting.

1. Stock Solutions
<ol>
	<li>Resuspend the lyophilized active motif powder in sterile H2O to a final concentration of 2 &#181;g/&#181;l. Tap carefully for mixing.</li>
	<li>Prepare small aliquots (e.g., 12 &#181;l each, 2 &#181;l are required per reaction) and store them at -20 &#176;C.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 &#181;l growth medium including antibiotics. 
	NOTE: ...

<ol>
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Here, we present a protocol to study the significance of protein expression levels in controlling cellular functions in a dose driven manner. The protocol described allows for a fine-tuned manipulation of protein expression in various mammalian cell types, including hippocampal neurons, hence facilitating detailed studies of protein function on the cellular level.
Although RNA interference represents a well known strategy to down-regulate POIs, it has its disadvantages (cp. 14). Not...

A tight control of protein expression is essential for every living organism to command its own development as well as to react to environmental signals. Hence, a multitude of mechanisms has been invented during evolution to precisely regulate the expression level of each protein encoded by the app. 20,000 genes existing in any eukaryotic cell at any given time of its life. Taking place at different stages of protein production, regulatory mechanisms range from the management of chromatin structure, transcription and RNA handling to the direction of posttranslational protein modifications, transport and degradation.
It is therefore not surprising that malfunctions in the underlying molecular machineries and altered protein expression levels have been associated with diverse diseases such as cancer or intellectual disabilities. Indeed, looking at the outstanding complexity of neuronal development and mammalian brain function, the sensitivity of these sophisticated systems to alterations in protein expression manifests itself in several well-known intellectual deficits including Alzheimer and Parkinson disease (AD and PD) as well as autism spectrum disorders (ASD) like the Fragile X Syndrome (FXS). The latter disease is characterized by an extensive misexpression of a variety of proteins, which is due to the loss of a single translation regulating protein, FMRP (Fragile X Mental Retardation Protein)1-4. Furthermore, chromosomal rearrangements affecting the Variable charge x linked protein A (VCXA), a protein, that manages mRNA stability and translation by modifying mRNA capping5, have recently been associated with intellectual deficits, while point mutations were not identified in patients with cognitive disabilities as of now6,7, suggesting that the observed mental impairments originate from altered VCXA expression and dysregulated expression of its target proteins. In line with these findings, a study investigating whether de novo copy number variations of genes are associated with ASD established that novel gene duplications and deletions are a significant risk factor for ASD8, thus supporting the idea that elevated or diminished protein expression levels may cause intellectual deficits.
Remarkably, recent research further provided evidence that the expression level of a given protein is precisely adjusted to prevent its aggregation as a consequence of high protein amounts with almost no safety margins9. It has therefore been proposed that even small increments are sufficient to induce diseases such as AD and PD9. Although the variety of molecular machineries contributing to protein expression control suggests a complex regulation scheme in the light of these findings, a study investigating the expression level of over 5,000 mammalian genes10 demonstrated that nature preferred a more parsimonious scheme: The cellular abundance of proteins was shown to be predominantly regulated at the level of translation10, thus illustrating that the management of RNA availability mainly serves to fine-tune protein expression.
Studying the dose of proteins of interest (POIs) is therefore not only important to the understanding of the endogenous functions of a protein, but also to the investigation of many diseases and the development of therapies. Thus, last decades have seen the advance of several strategies using RNA interference to manipulate POI dosage. Though RNA interference is widely used to study protein function and is even being applied in clinical trials to treat cancer or ocular diseases as well as to pursue antiviral therapies in patients11-13, some difficulties may arise which might render the strategy impossible. For example, the seed sequence, which drives the knockdown by homology is comparable short, hence promoting off-target effects. Since highly efficient sequences are rare and need to be found among thousands of options (reviewed in 14), identifying the right sequence can be time-consuming and costly, but results may still be disappointing.
An alternative strategy is to directly target the POI by antibodies. Here, we illustrate the use of the protein carrier Chariot&#160;(manufactured by Active Motif) to reduce cellular protein availability, and the employment of three-dimensional reconstructions to study protein function following knock-down.
The active motif of Chariot, itself a 2.8 kDa peptide, is used to shuttle peptides, proteins and antibodies across membranes of mammalian cells15. The peptide associates with POIs by forming non-covalently coupled macromolecular complexes utilizing hydrophobic interactions, whereupon Chariot-POI complexes are internalized into cells in an endosome-independent manner. Importantly, Chariot was indicated to neither affect the intracellular localization of shuttled proteins, nor to exert cytotoxic effects or to affect the biological activity of its cargo15.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

In the following paragraphs, exemplary results illustrating a functional knockdown of a POI (Simiate, for further details please see 16,17) using Chariot reagent and antibody interference are presented. The findings demonstrate that diminished expression of Simiate impairs transcriptional activity, and, in a dosage dependent manner, induces apoptosis, culminating in mortality rates of over 99% if higher antibody amounts (&#62; 1 &#181;g antibody) are applied (these discoveries were publis...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

The overall goal of the following experiment is to study protein functions and the role of altered protein expression in an efficient and tightly controlled manner. This is achieved by applying antibodies to reduce the amount of functional protein available to the cell. To accomplish this, antibodies are coupled to a peptide named Chariot, which mediates a passage across cellular membranes.Next, cellular structures of interest are imaged and 3D-reconstructed in order to evaluate effects of the treatment. The results reveal that Chariot peptides are particularly well-suited to target nuclear proteins by antibody interference. The main advantages of this technique over existing methods like RNA interference are that the antibodies shuttled into the cell are effective immediately, and that the researcher may strictly control the degree of interference by adjusting antibody dosages.Though this method can provide insight into protein functions in many cell types, it is particularly interesting for targeting proteins in primary neuronal cultures, which are very sensitive to treatments but tolerate Chariot very well. In preparation, resuspend lyophilized Chariot peptide powder in sterile water at two micrograms per microliter. Tap the tube carefully to mix the powder into solution.Then prepare small working aliquots, just two microliters are needed for each reaction, and store the aliquots at 20 degrees Celsius. Next, prepare mammalian cells for transfection by seeding them to a 24-well plate with half a milliliter of medium containing antibiotics. When using neurons, seed the cells only in the two central rows and at a low density.The empty wells are useful later. Be quick. Cultured neurons should not be out of the incubator for more than a few minutes.Until the neurons are at the desired developmental stage, exchange about one quarter to one half of their medium twice a week. Non-neuronal cultures should be used when the cells are at 50%confluence. The following procedure is shown for six tests and six control samples.For each cargo, prepare a dilution of 0.1 to 2 micrograms in 50 microliters of 1X PBS per sample. Macromolecular complexes of pre-bound primary and secondary antibodies can be used as cargo as well. Mix the solution with a gentle vortex, followed by a brief centrifugation.Then dilute two microliters of Chariot stock solution in 50 microliters of PBS for each reaction. Use separate tubes for every sample in order to prevent self-association of Chariot. Now, combine the prepared proteins in the Chariot dilutions.Mix and spin the combined proteins. Incubate the sample for 30 minutes at room temperature to allow for the formation of Chariot cargo complexes. The optimal protein peptide amount depends on the cargo and the cell type used.Therefore, this amount should be determined empirically. In advance, pre-warm some growth medium with no additives as well as 1X PBS containing 0.5 millimolar magnesium chloride and calcium chloride to 37 degrees Celsius. Now, to begin, dilute the prepared Chariot complexes with 100 microliters of medium.Remove the medium from the plated cells and transfer to the empty wells for reuse. Then wash the cells gently with 500 microliters of pre-warmed 1X PBS, calcium, magnesium chloride solution. Apply the medium carrying the Chariot complexes to the cells and rock the plate gently to ensure an even distribution of the solution.Quickly return the cells to the incubator and wait an hour before preceding. For neurons, after an hour of incubation, transfer the saved media to the wells. For all other cells, add serum to a final concentration of 10%Cultured neurons are very sensitive, such as to changes in media temperature and pH.Therefore, these cells should be washed and transfected gently with pre-warmed solutions and returned to the incubator as soon as possible. Continue the incubation for as long as needed, depending on the transfected cargo and the cell type. After the incubation, process the cells for subsequent analyses as usual.Fixation protocols, as well as live imaging, are both compatible with this technique. Using a laser scanning microscope with a 63X lens, take images in a distance of approximately 0.25 to 0.5 micron in order to obtain Z-stacks of sufficient detail while avoiding bleaching. The precise layer distance depends on the size of the structure to be reconstructed and needs to be determined individually.For image reconstruction, open the LSM files in Imaris. With this software, the Escape button is used to switch between Select and Navigate. Also, multiple objects can be selected by holding down the Control key.Using the Display Adjustment, go into each channel and adjust the contrast until the brightest structures reach saturation. This adjustment does not influence the surface construction but it does assist in thresholding the image. If the object to reconstructed is contained in different files, use the Edit and Add Slices function to combine the corresponding files.Next, click on the Add New Surfaces icon and select Segment only a Region of Interest. Now, adjust the margins of the selection box to fit the object of interest. Adjust the shape and tilt of the rectangular shape until all the required structures are within the box.The selection needs to be adjusted in each image all through the Z-plane. Undesired objects may be removed later on. Next, select an appropriate channel.To proceed, use the thresholding tool to reconstruct the structure of interest. The surface area detail level, or sphere diameter, should be set to match the structures being reconstructed. Depending on the signal characteristics, either absolute intensity or local contrast can be used.Generally, local contrast works better for diffuse signals. Due to the characteristics of this signal, local contrast is used to reconstruct the protein inside the nucleus. In any case, the settings needs to be adjusted separately for each signal or structure of interest.Complete the reconstruction by clicking on the green arrow at the bottom of the screen. Then adjust the object further using the Pencil tool. Carefully compare the reconstructions to the original signal, and cut and remove surfaces by tilting the images as needed so the Cut tool can accurately serve its purpose.Now, measure the volume, area, and intensities of each surface by selecting Statistics, Detailed statistics, and Average Values. If desired, color-coded statistics can be produced by selecting the Color tab of the corresponding surface and changing the selected option from Base to Statistic Coded. Employing the Take Snapshot tool, pictures can be taken for publication.If several objects within the same region are to be reconstructed, the Store Parameters function can be used to save the information. Simiate expressed in HEK293 cells was specifically detected by Simiate antibodies shuttled into the cells by Chariot peptides. Simiate antibody assemblages enter the soma as well as the nucleus, following application, where they co-localize specifically with FLAG-Simiate as marked by the yellow arrows.In a dose-dependent manner, Simiate antibodies induced massive apoptosis in HEK293 cells while control antibodies had no effect. Given that neuronal cells are very sensitive to toxic effects, the technique was applied to primary hippocampal cultures where there was no effect on the viability of neurons when comparing mock, or untreated, and Chariot-treated cells. Following the same application scheme, goat anti-guinea pig Alexa568 antibody assemblages were seen in about 83%of the cells where they occurred in somata as well as nuclei to varying extents.These observations were further supported by a 3D analysis, which was consistent with the results from the HEK293 cells. Hence, the Chariot technique can be used successfully to transfect dissociated primary hippocampal neurons. Once mastered, this technique can be done in three to six hours if performed properly.The complex formation of Chariot and the protein of interest takes about 40 minutes. To transfect the cells with these complexes, it will take 70 minutes and additional one to four hours depending on the size and properties of the cargo. Following this procedure, other methods can be performed in order to analyze the function of a protein in more detail, such as live imaging and motility assays, analysis and electrophysiological recordings.

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