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

Summary

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.

Abstract

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  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.

Introduction

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 now^6,7, 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 patients^11-13, 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 (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.

Protocol

1. Stock Solutions

1. Resuspend the lyophilized active motif powder in sterile H₂O to a final concentration of 2 µg/µl. Tap carefully for mixing.
2. Prepare small aliquots (e.g., 12 µl each, 2 µl are required per reaction) and store them at -20 °C.

2. Preparation of Cells

1. Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 µl growth medium including antibiotics.
   NOTE: When using neurons, seed the cells at low density and use only the two middle rows of the 24 well plate. Each well will thus have an empty counterpart to harbor the medium during incubation (step 4.2). When handling neurons, always make sure that the cells are not kept outside the incubator for more than a few minutes.
2. Culture the cells at appropriate conditions (humidified, 5% CO₂ and 37 °C) until the cells are app. 50% confluent.
   NOTE: When using neurons, culture the cells until the desired developmental stage is reached and change app. 25-50% of the medium twice a week. Medium: Neurobasal medium (containing 1x B27, 5 mM L-glutamine and 1x Penicillin and Streptomycin; compare with ref 16).

3. Chariot Complex Formation

1. Per reaction, dilute 0.1-2 µg of the POI or the corresponding antibody as well as the control protein, or control antibody, respectively, in 50 µl of PBS.
   NOTE: The technique also works with macromolecular complexes consisting of pre-bound primary and secondary antibodies (cp. 'Representative Results', Figure 1). Mix and spin.
2. For each sample, dilute 2 µl Chariot stock solution in 50 µl of PBS using separate tubes in order to avoid self-association of Chariot. Mix and spin.
3. Transfer the diluted POI or antibody (step 3.1) to the Chariot dilution by pipetting. Mix and spin.
4. Incubate the mixture for 30 min at room temperature (Chariot-POI complexes will form).

4. Transfection of Cells

1. Dilute the Chariot complexes (step 3.4) in 100 µl pre-warmed growth medium (37 °C, no additives). Remove the medium and wash the cells once using pre-warmed 1x PBS.
   NOTE: When using neurons, do not discard the medium, it will be reused. Keep it at 37 °C. For washing, use PBS containing 0.5 mM MgCl₂ and CaCl₂.
   Suggestion: keep the medium in empty wells while incubating the plate (step 4.5).
2. Apply the Chariot complex solution (step 4.1) to the cells and rock the cells gently to ensure an even distribution of the solution. Grow the cells under standard conditions (step 2.2) for 1 hr. Add serum to a final concentration of 10%. When using neurons, add the medium from step 4.1. Grow the cells for 1 to 2 more hours.
   NOTE: The optimal incubation time depends on the size and properties of the cargo and may need to be adjusted empirically. The following suggestions may serve as a guideline: For peptides, 1 hr is usually sufficient, for proteins 1-2 hr are recommended, for antibodies 2 hr, and for the transfection of neurons with antibodies 4 hr.
3. Process cells for analysis as usual. Please note: the technique is compatible with experiments using fixation protocols as well as live imaging.

5. Imaging

1. Using a laser scanning microscope, take z-stacks of cells and/or cell compartments of interest using approximately 0.25-0.5 µm distance. The precise layer distance depends on the size of the structure to be reconstructed and needs to be determined individually.

6. Reconstruction

1. In the following steps, use the Esc bottom to switch between Select and Navigate, Cntr to select multiple objects by clicking on them and shift key to cut objects (cp. step 6.10).
2. Open the lsm-file in Imaris.
3. Using the Display Adjustment for each channel, contrast the picture until the brightest structures reach saturation. Please note that this adjustment will not influence the surface construction, it only serves to assist the experimenter in thresholding the image.
4. Click on the Add new Surfaces icon. A wizard will open.
5. Select: Segment only a Region of Interest. Proceed to the next step.
6. Adjust the margins of the selection box to fit your object of interest. Please bear in mind that the image has 3 dimensions and that the selection needs to be adjusted at the z plane as well. If the rectangular shape of the selection box should not match the object of interest properly, turn the object and/or enlarge the box until all required structures are embedded. Undesired objects may be removed later on (cp. step 6.10). Proceed.
7. Select the appropriate channel. 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. In any case, the settings need to be adjusted separately for each signal or structure of interest, respectively.
8. Using the Thresholding tool, reconstruct the structure of interest.
9. Complete the reconstruction by clicking on the green arrow bottom.
10. Adjust the object further by using the pencil tool to cut and remove surfaces as required. It is only possible to cut in north-south direction, therefore, tilt the image in order to cut the desired object.
11. Obtain measurements of volume, area and intensities for each surface by selecting Statistics, Detailed Statistics, and Average Values.
12. (Optional step) To observe color-coded statistics (cp. Figure 2E and F), select the color tab of the corresponding surface and mark 'Statistic Coded' instead of 'Base'. Several options will be displayed.

Results

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 (> 1 µg antibody) are applied (these discoveries were published pr...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chariot	Active Motif	30025	store at -20 °C
Neurobasal medium	Life technologies	21103-049	warm up to 37 °C before using
1x B27	Life technologies	17504044	store at -20 °C
L-glutamine	Life technologies	25030-149	store at -20 °C
Penicillin and Streptomycin	Life technologies	15140-122	store at -20 °C
Imaris software	Bitplane	n.a.	expensive, but unmatched
Laser Scanning Microscope	Zeiss	n.a.