Name: demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay
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This work was supported by grant no. 2016/23/D/NZ3/01314 from the National Science Centre (NCN), Krakow, Poland.

1. Generation of expression plasmids
<ol>
	<li>Examine membrane topology of the proteins of interest using a topology predicting tool.</li>
	<li>Design the cloning strategy so that the larger and smaller fragments would face the cytoplasm once the fusion proteins have been inserted into Golgi membranes. If, as in the case&#160;presented here, both N- and C-termini of the proteins of interest are cytoplasmically oriented, tag the proteins in eight possible ways (see Figure 1B). If N- or C-terminus of one or both proteins of interest is luminally oriented, exclude it from tagging.</li>
	<li>Subclone the genes of interest into appropriate expression vectors (see Table of Materials) by following standard cloning protocols. 
	NOTE: Testing all the possible orientations is recommended, since some tagging options may not work due to insufficient proximity, suboptimal orientation, or spatial constraints.</li>
</ol>
2. Transient transfection of the expression plasmids into the cells
<ol>
	<li>Harvest the adherent HEK293T cell culture by trypsinization and resuspend the cells in a dedicated complete growth medium. Plate the cells (2 x 104/100 &#181;L/well) onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls including replicates. 
	NOTE: Attempt to use only the inner 60 wells of the plate to minimize thermal shifts and avoid overnight evaporation. Using poly-D-lysine-coated plates is highly recommended such as the ones indicated in the Table of Materials, otherwise cells may detach during the subsequent washing steps.</li>
	<li>Culture the cells overnight in standard conditions (37 &#176;C, 5% CO2).</li>
	<li>On the next day transfect the cells with the desired combinations of expression plasmids obtained in point 1.1.
	<ol>
		<li>Dilute expression plasmids in a serum-free medium (see Table of Materials) to 6.25 ng/&#181;L for each construct.</li>
		<li>Add the lipid-based transfection reagent at an appropriate lipid-to-DNA ratio and incubate according to the manufacturer&#8217;s instruction.</li>
		<li>Add 8 &#181;L of lipid:DNA mixture to designated wells. Mix the content of the plate by gentle rotation. This results in transfection of both expression constructs at 50 ng/well.</li>
	</ol>
	</li>
	<li>Culture the cells for 20-24 h in standard conditions (37 &#176;C, 5% CO2). 
	NOTE: Culturing the cells for a longer time may result in higher levels of fusion protein expression, which may promote a non-specific association between the fragments.</li>
</ol>
3. Medium exchange
<ol>
	<li>On the next day replace the conditioned medium with 100 &#181;L of a serum-free medium in each well. Make sure that the cells have not detached upon medium exchange. 
	NOTE: This step should be done 2-3 h before the addition of the furimazine working solution. Serum withdrawal minimizes background caused by autoluminescence of furimazine.</li>
</ol>
4. Preparation of furimazine working solution
<ol>
	<li>Just before the measurement, mix 1 volume of furimazine with 19 volumes of a dilution buffer (a 20-fold dilution). 
	NOTE: The total volume of the furimazine working solution to be prepared depends on the number of individual wells to be analyzed (the furimazine working solution is added to the cell culture medium in a 1:5 ratio, therefore, to each well previously filled with 100 &#181;L of a serum-free medium 25 &#181;L of the furimazine working solution should be added).</li>
	<li>Add the furimazine working solution to designated wells (25 &#181;L/well). Gently mix the plate by hand or using an orbital shaker (e.g., 15 s at 300-500 rpm).</li>
</ol>
5. Measuring luminescence
<ol>
	<li>Insert the plate into a luminescence microplate reader.
	<ol>
		<li>For experiments that are to be performed at 37 &#176;C equilibrate the plate for 10-15 min at the indicated temperature.</li>
	</ol>
	</li>
	<li>Select the wells to be analyzed.</li>
	<li>Read luminescence with integration time of 0.3 s. Continue to monitor luminescence for up to 2 h when required.</li>
</ol>
6. Data analysis
<ol>
	<li>Calculate mean values and standard deviations for all the tested and control combinations.</li>
	<li>Analyze data using one-way ANOVA with multiple comparisons.</li>
	<li>Calculate fold change values by dividing a mean luminescence obtained for combinations of interest by a mean luminescence obtained for the corresponding negative controls. Evaluate the results. 
	NOTE: The approach to data analysis proposed here assumes that the interaction can be claimed if the luminescence obtained for the tested combination is statistically significantly higher than the luminescence obtained for the corresponding control combination and, at the same time, the ratio of these two values exceeds 10.</li>
</ol>

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The authors have nothing to disclose.

Here we provide a detailed protocol enabling the demonstration of heterologous complexes formed between Golgi-resident type III membrane proteins, such as NSTs, using the split luciferase complementation assay. The proposed approach to data analysis and interpretation involves relating the luminescence obtained for the protein combination of interest to the luminescence obtained for the corresponding control combination, which is composed of one of the proteins of interest fused with the larger fragment and the control protein of a bacterial origin fused with the smaller fragment. The latter is expressed in the cytoplasm of mammalian cells;&#160;therefore, using it as a reference requires that N- and/or C-termini of the proteins, whose interaction is to be analyzed, are on the cytoplasmic side of the Golgi membrane.
The critical steps in the protocol are plasmid design, plating the cells to be transfected, transfection itself, the medium exchange, preparation of the furimazine working solution and adding it to the cells. The plasmid design should be made in such a way that both luciferase fragments are facing the cytoplasm, otherwise control combinations will not work and the reference RLU values will be missing. Cells to be transfected should be plated as specified in the protocol, otherwise the transfection efficiency might be suboptimal. We recommend using multiwell plates with the poly-L-lysine-coated surface to support cell attachment while the medium is being exchanged. Finally, the furimazine working solution should be freshly prepared and immediately added to the cells. Once it is added, the readout should be performed as soon as possible.
To avoid inconclusive results, positive and negative controls should always be included. The positive control should be always employed so that the transfection efficiency is monitored. It is very important that all the possible fusion proteins that are topologically compatible with each other as well as with the HaloTag-based negative control are generated and combined. If possible, the negative control comprising of a membrane protein non-interacting with any of the proteins of interest should be used. Here, we did not employ such a control, as its development is still on the way. Instead, we forced a specific disassembly of the complexes of interest by co-expressing an extra, untagged copy of one of the analyzed proteins. The expression vectors we used carry the relatively weak herpes simplex virus-thymidine kinase (HSV-TK) promoter, which ensures low expression levels that are optimal for obtaining specific outcome. However, in the case of suboptimal results it might be beneficial to use the CMV-based vectors or, alternatively, optimizing the amount of plasmids used for transfection.
The presented method, although powerful and convenient, has some limitations. First, this method does not allow to conclude whether the identified complexes correspond to dimers or higher-order oligomers. Besides, the subcellular localization of the interacting proteins cannot be monitored. This is, however, possible upon the extension of the basic protocol with bioluminescent imaging, although spatial resolution of such images would be significantly lower when compared with fluorescence-based approaches.
The presented method is very fast and efficient (the measurement takes up to several minutes and the data are obtained from thousands of cells). Data processing and interpretation is also relatively straightforward. Little or no optimization of the basic protocol is needed. The only specific equipment required is a luminometer. The split luciferase complementation assay and BiFC work on the same essential principle, i.e., reconstitution of a functional protein from its non-functional fragments. General advantages of bioluminescence-based approaches over the ones based on fluorescence were already listed in the Introduction. A specific advantage of the split luciferase complementation assay over BiFC is that the former is fully reversible15. In BiFC, once a fluorescent protein is reconstituted, it would not dissociate back into the corresponding non-fluorescent fragments17. In contrast, the assembly of the NanoLuc subunits is reversible, which creates a unique opportunity to study the dynamics of PPIs. Finally, the exceptional sensitivity of the presented method allows to assume that this approach should work even with the cell lines that are difficult to transfect.
The protocol presented here allows to determine whether the two Golgi-resident type III membrane proteins interact. As already mentioned, the basic setup of the method can be coupled with bioluminescence imaging to confirm the subcellular localization of the PPI of interest. The reversibility of this split luciferase complementation assay allows to study the dynamics of PPIs in real time. The luminescent signal derived from furimazine is sustained for about 2 hours. However, substrates for the split luciferase complementation assay ensuring a substantially longer (hours to days) duration of the resulting signal are also available. This method allows for the identification of the factors that trigger or prevent the PPI of interest. Some of the most recent examples of its application include studies on interactions between G proteins and G-protein-coupled receptors18, protein conformational changes19, protein ubiquitination20, internalization of cell surface receptors21, and identification of factors that modulate PPIs22,23. Therefore, this method appears to be a versatile tool with a high potential to fulfill even very challenging experimental goals.

This manuscript describes a step-by-step protocol to check for the presence of heterologous interactions between Golgi-resident type III membrane proteins in transiently transfected human cells using the most recent variant of the split luciferase complementation assay. The procedure has been most extensively tested against nucleotide sugar transporters (NSTs) but we were also able to obtain positive results for other Golgi-resident type III membrane proteins whose N- and/or C-termini are facing the cytoplasm.
Our research group explores the role of NSTs in glycosylation of macromolecules. NSTs are Golgi- and/or ER-resident type III membrane proteins with N- and C-termini facing the cytoplasmic side of the organellar membrane1. NSTs are thought to carry nucleotide-activated sugars across organelle membranes to supply glycosyltransferases with their substrates. NSTs form dimers&#160;and/or higher oligomers2,3,4,5,6,7,8,9,10. Moreover, heterologous interactions between&#160;different NSTs have also been reported6,11. NSTs were also demonstrated to form complexes with functionally related glycosylation enzymes12,13,14. We sought for an alternative to the presently used technique, fluorescence lifetime imaging (FLIM)-based FRET approach, for studying interactions of NSTs and functionally related Golgi-resident proteins, so we decided to test the split&#160;luciferase complementation assay. It allowed us to identify a novel interaction between an NST and a functionally related glycosylation enzyme9.
The most recent modification of the split luciferase complementation assay, NanoBiT, is used in the protocol presented here15. It relies on the reconstitution of the luciferase enzyme (e.g., NanoLuc) from the two fragments - the large one, termed as large BiT or LgBiT, a 17.6 kDa protein, and the small one, composed of only 11 amino acids, termed as small BiT or SmBiT. The two proteins of interest are fused with the complementary fragments and transiently expressed in a human cell line. If the two fusion proteins interact, a luminescence is produced in situ upon addition of a cell-permeable substrate. These two fragments have been optimized so that they associate with minimum affinity unless being brought together by an interaction between the proteins of interest they are fused to.
In general, bioluminescence-based methods have some advantages over the ones based on fluorescence. Bioluminescent signals have a higher signal-to-noise ratio because the background luminescence is negligible compared to the luciferase-derived signal16. In contrast, fluorescence-based approaches usually suffer from a relatively high background caused by the phenomenon of autofluorescence. Besides, bioluminescence is less detrimental to the analyzed cells than fluorescence, as in the former case there is no need to excite the sample. For those reasons bioluminescent approaches to studying PPIs in vivo outcompete the commonly used fluorescent methods like F&#246;rster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC).
Our protocol relies on referring luminescence obtained for the protein combination of interest to luminescence obtained for the control combination. The latter includes the one of the tested proteins which is fused with a larger fragment and a control&#160;protein (e.g., HaloTag),&#160;fused with a smaller fragment. The latter&#160;is a protein of bacterial origin that is not expected to interact with any of mammalian proteins. Using this protein as a control poses limitations to the topology of the Golgi-resident pairs of proteins to be analyzed. Since in mammalian cells this protein is synthesized in the cytoplasm, both proteins of interest should have at least one cytoplasmic tail.
This approach can be particularly useful for initial screening of PPIs. It may become the method of choice when the fusion proteins of interest are expressed at levels that are simply insufficient for other approaches to be applied. Similarly, the split luciferase complementation assay can be the best option if the proteins of interest are expressed at high levels, but this adversely affects their subcellular localization or is known to force non-specific interactions. Since the smaller fragment has only 11 amino acids, the split luciferase complementation assay can be applied when using larger tags is impossible. Finally, it can be employed to further confirm data obtained using other techniques, as in the case&#160;presented here.

<table><tbody><tr><td>0.25% trypsin-EDTA solution</td><td></td><td></td><td></td></tr><tr><td>Adherent mammalian cell line</td><td></td><td></td><td></td></tr><tr><td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td><td>Corning</td><td>356651</td><td></td></tr><tr><td>Cell culture centrifuge</td><td></td><td></td><td></td></tr><tr><td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td><td></td><td></td><td></td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td><td>Promega</td><td>GM3000</td><td></td></tr><tr><td>Growth medium dedicated to the cell line used</td><td></td><td></td><td></td></tr><tr><td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td><td></td><td></td><td></td></tr><tr><td>NanoBiT MCS Starter System</td><td>Promega</td><td>N2014</td><td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td></tr><tr><td>Nano-Glo Live Cell Assay System</td><td>Promega</td><td>N2011</td><td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td></tr><tr><td>Opti-MEM I Reduced Serum Medium, no phenol red</td><td>Gibco</td><td>11058021</td><td></td></tr><tr><td>Oribital shaker</td><td></td><td></td><td></td></tr><tr><td>Software for data analysis (e.g. GraphPad Prism)</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td></td><td></td><td></td></tr></tbody></table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions between&#160;different NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. The&#160;luciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

To obtain the most reliable data in this approach all the possible combinations should be tested (see Figure 1). In parallel, positive and negative controls should be included. The positive control should consist of the two proteins that are known to interact, of which one is fused with the larger fragment and the other is fused with the smaller fragment. The negative control ideally should consist of the two non-interacting type III membrane proteins tagged likewise. However, establishing such a control may be challenging, as the lack of interaction of the two control proteins should be thoroughly confirmed using several alternative approaches. Therefore, one might employ a cytosolic protein of non-human origin that is not expected to interact with any human protein (e.g., HaloTag) as a negative control, if N- and/or C-termini of the proteins, whose interaction is to be determined, are cytoplasmically exposed as in the case presented here. We highly recommend an additional verification of the results obtained using this type of control by co-expression of the&#160;untagged variants of either of the proteins of interest combined with titration of the corresponding plasmids. If the two proteins specifically interact, an extra copy of either of them lacking the luciferase fragment should result in a specific decrease of luminescence.
Relative luminescence units (RLU) values typical of positive and negative combinations are listed in Table 1. The results were obtained for the two NSTs, SLC35A2 and SLC35A3, that were shown to associate by co-immunoprecipitation and FLIM-FRET6 as well as in situ proximity ligation assay11. Both SLC35A2 and SLC35A3 are Golgi-resident type III membrane proteins with N- and C-termini facing the cytoplasm (Figure 1A). Therefore, there are eight possible tagging options and the resulting fusion proteins can be set together also in eight possible ways (Figure 1B). Mean RLU values corresponding to all the tested and control combinations are depicted in Figure 2A. The positive control is also included. An initial requirement for a selected result to be considered indicative of an interaction is that the RLU value obtained for the combination of interest is statistically significantly higher than the RLU value obtained for the corresponding control combination. In Figure 2A three such combinations are seen (LgBiT-SLC35A2 + SLC35A3-SmBiT, SmBiT-SLC35A2 + LgBiT-SLC35A3 and SLC35A2-SmBiT + LgBiT-SLC35A3). The next step in the analysis of results involves obtaining ratio values for the combinations of interest by dividing the corresponding RLU values by RLU values obtained for the respective controls. The results of such analysis are shown in Figure 2B. According to the manufacturer&#8217;s suggestions, ratios between 10 and 1000 are highly indicative of specific interactions. There are two combinations that meet these criteria (SmBiT-SLC35A2 + LgBiT-SLC35A3 and SLC35A2-SmBiT + LgBiT-SLC35A3) and support the idea that SLC35A2 and SLC35A3 interact. However, the fact that the other six combinations are negative does not mean that there are no interactions at all between the respective fusion proteins, but rather suggests that the tagging strategy was not optimal in these cases. This example shows how important it is to test all the possible combinations.
Data presented in Figure 2 were additionally confirmed by the&#160;co-expression of HA-tagged SLC35A2 or SLC35A3 with the combination that resulted in the highest relative luminescence, namely SLC35A2-LgBiT + SmBiT-SLC35A3. Varying amounts of the plasmids encoding HA-tagged NST variants were used for co-transfection. This resulted in a statistically significant, dose-dependent decrease in RLU values (Figure 3). Specific and dose-dependent disruption of the interaction between SLC35A2-LgBiT and SmBiT-SLC35A3 by the simultaneous co-expression of HA-tagged NST variants is shown in Table 2.
The most common&#160;problem associated with the&#160;method presented here&#160;is the poor efficiency of transfection. To verify&#160;whether this is the cause of suboptimal results, include positive control in all experiments. The positive control we employed to obtain the data&#160;presented here consists of the two interacting fusion proteins: cAPM-dependent protein kinase catalytic subunit alpha (PRKACA) fused with the smaller fragment and cAPM-dependent protein kinase type II-alpha regulatory subunit (PRKAR2A) fused with the larger fragment. In our hands, the corresponding RLU value reached ~6 x 105. A significant (2-3 orders of magnitude) drop in this number might be indicative of the suboptimal efficiency of the performed transfection. In such a case we recommend checking on the well-being of the cultured cells and making sure that the appropriate number of cells is being plated for transfection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61669/61669fig1v3.jpg" /> 
Figure 1: Schematics of membrane topology and tagging.&#160;(A) Membrane topology of SLC35A2 and SLC35A3 proteins. (B) Possibilities of tagging SLC35A2 and SLC35A3 proteins with the split luciferase complementation assay fragments and combining the resulting fusion proteins for the assay. <a href="https://www.jove.com/files/ftp_upload/61669/61669fig1v3large.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/61669/61669fig2v3.jpg" /> 
Figure 2: Results of the split luciferase complementation&#160;assay performed in HEK293T cells for the selected protein combinations and the corresponding negative controls (processed data).&#160;(A) RLU values obtained for the selected protein combinations and the corresponding negative controls. Negative, HaloTag tagged with SmBiT; PRKAR2A, cAPM-dependent protein kinase type II-alpha regulatory subunit; PRKACA, cAPM-dependent protein kinase catalytic subunit alpha. Data were analyzed using one-way ANOVA with multiple comparisons and are presented as a mean &#177; standard deviation (SD) from three technical replicates. p &#60; 0.1 *, p &#60; 0.05 **, p &#60; 0.01 ***. (B) Fold changes calculated by dividing a mean luminescence obtained for the tested combination (RLU SAMPLE) by a mean luminescence obtained for the corresponding negative control (RLU CONTROL). Threshold value considered indicative of an interaction was set at 10. <a href="https://www.jove.com/files/ftp_upload/61669/61669fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61669/61669fig3v3.jpg" /> 
Figure 3: Specific and dose-dependent disruption of the interaction between SLC35A2-LgBiT and SmBiT-SLC35A3 by simultaneous co-expression of HA-tagged NST variants.&#160;HEK293T cells were transfected with plasmids encoding SLC35A2-LgBiT and SmBiT-SLC35A3 and, additionally, either with an empty pSelect plasmid (mock) or with increasing amounts of pSelect plasmids encoding HA-tagged SLC35A2 (left panel) and SLC35A3 (right panel). Data were analyzed using one-way ANOVA with multiple comparisons and are presented as a mean &#177; standard deviation (SD) from three technical replicates. p &#60; 0.05 **, p &#60; 0.001 ****. <a href="https://www.jove.com/files/ftp_upload/61669/61669fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Results of the assay performed in HEK293T cells for the selected protein combinations and the corresponding negative controls (raw data). Unprocessed RLU values obtained for the selected protein combinations and the corresponding negative controls are shown. Negative, HaloTag tagged with SmBiT; PRKAR2A, cAPM-dependent protein kinase type II-alpha regulatory subunit; PRKACA, cAPM-dependent protein kinase catalytic subunit alpha, TR, technical replicate. <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_1.xlsx" target="_blank">Please click here to download this table.</a>
Table 2: Specific and dose-dependent disruption of the interaction between SLC35A2-LgBiT and SmBiT-SLC35A3 by simultaneous co-expression of HA-tagged NST variants (raw data). Unprocessed RLU values obtained for the selected protein combinations are shown. Mock, cells expressing SLC35A2-LgBiT and SmBiT-SLC35A3 co-transfected with an empty pSelect vector; HA-SLC35A2, cells expressing SLC35A2-LgBiT and SmBiT-SLC35A3 co-transfected with the indicated amounts of a pSelect plasmid encoding SLC35A2 tagged with the HA epitope at the N-terminus; HA-SLC35A3, cells expressing SLC35A2-LgBiT and SmBiT-SLC35A3 co-transfected with the indicated amounts of a pSelect plasmid encoding SLC35A3 tagged with the HA epitope at the N-terminus; TR, technical replicate. <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_2.xlsx" target="_blank">Please click here to download this table.</a>

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Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

The&#160;protocol presented here is intended to demonstrate the occurrence of heterologous interactions between Golgi-resident type III membrane proteins with cytoplasmically exposed N- and/or C-termini in live mammalian cells using the most recent variant of the split luciferase complementation assay.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

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Nanyang Technological University

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JoVE Journal

Biology

2.3K Views.  University of Wroclaw. The protocol presented here is intended to demonstrate the occurrence of heterologous interactions between Golgi-resident type III membrane proteins with cytoplasmically exposed N- and/or C-termini in live mammalian cells using the most recent variant of the split luciferase complementation assay.

Video: Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

Cancer Research

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Biochemistry

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Developmental Biology

A Simple Assay to Measure ER-Mitochondria Contacts for High-Throughput Screens

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts &#8212; Implications for Neurodegenerative Disease Pathogenesis

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

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, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

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

The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

Source: Rezaei, R., et al. <a href="https://app.jove.com/v/62488">Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021).
The video demonstrates the luciferase assay used to quantify luciferase-tagged proteins. The assay uses an engineered luciferase enzyme composed of a large fragment that binds with the small fragment, which is conjugated with a protein of interest. The bound subunits form an active enzyme that, in the presence of the substrate, releases a luminescent signal. This signal is then quantified using a luminometer.

Source: Rezaei, R., et al. Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021).
The video ...

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Summary

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Chapters in this video

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

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

The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters