Name: In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay (Video) | JoVE Journal
Uploaded: 2020-05-05T14:00:00.000+00:00
Duration: 8 min 56 s
Description: Learn proximity ligation assay for detecting protein-protein interactions in situ using C. elegans germline. This method reveals spatial and temporal patterns to study developmental regulation.

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Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center funded by the NIH (P40OD010440). Confocal microscopy was performed in the University of Montana BioSpectroscopy Core Research Laboratory operated with support from NIH Awards P20GM103546 and S10OD021806. This work was supported in part by the NIH grant GM109053 to E.V., American Heart Association Fellowship 18PRE34070028 to X.W., and Montana Academy of Sciences award to X.W. The funders were not involved in study design or writing the report. We thank M. Ellenbecker for discussion.

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The authors have no conflicts of interest.

When studying PPIs in the C. elegans germline, the higher resolution offered by PLA compared to co-immunostaining allows visualization and quantification of locations where interactions occur in the germline. It was previously reported that DLC-1 directly interacts with OMA-1 using an in vitro GST pulldown assay26; however, this interaction was not recovered by an in vivo pulldown. The fluorescent co-immunostaining of 3xFLAG::DLC-1; OMA-1::GFP germlines shows an overlap in the ex...

Over 80% of proteins are estimated to have interactions with other molecules1, which emphasizes how important PPIs are to the execution of specific biological functions in the cell2. Some proteins function as hubs facilitating assembly of larger complexes that are necessary for cell survival1. These hubs mediate multiple PPIs and help organize proteins into a network that facilitates specific functions in a cell3. Formation of protein complexes is also affected by biological context, such as the presence or absence of specific interacting partners4, cell signaling events, and developmental stage of a cell.
C. elegans is commonly used as a model organism for a variety of studies, including development. The simple anatomy of this animal is comprised of several organs, including the gonad, gut, and transparent cuticle, which facilitates the analysis of worm development. The germline residing in the gonad is a great tool to study how germline stem cells mature into gametes5 that develop into embryos and eventually the next generation of progeny. The distal tip region of the germline contains a pool of self-renewing stem cells (Figure 1). As stem cells leave the niche, they progress into the meiotic pachytene and eventually develop into oocytes in the young adult stage (Figure 1). This program of development in the germline is tightly regulated through different mechanisms, including a post-transcriptional regulatory network facilitated by RNA-binding proteins (RBPs)6. PPIs are important for this regulatory activity, as RBPs associate with other cofactors to exert their functions.
There are several approaches that can be used to probe for PPIs in the worm, but each has unique limitations. In vivo immunoprecipitation (IP) can be used to isolate protein-protein complexes from whole worm extracts; however, this approach does not indicate where the PPI occurs in the worm. In addition, protein complexes that are transient and only form during a specific stage of development or in a limited number of cells can be difficult to recover by co-immunoprecipitation. Finally, IP experiments need to address the concerns of protein complex reassortment after lysis and non-specific retention of proteins on the affinity matrix.
Alternative approaches for in situ detection of PPIs are co-immunostaining, F&#246;rster resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC). Co-immunostaining relies on simultaneous detection of two proteins of interest in fixed worm tissue and measurement of the extent of signal colocalization. Use of super-resolution microscopy, which offers greater detail than standard microscopy7, helps to more stringently test protein colocalization beyond the diffraction-limited barrier of 200-300 nm8. However, co-immunostaining using both conventional and super-resolution microscopy works best for proteins with well-defined localization patterns. By contrast, it becomes much less informative for diffusely distributed interacting partners. Measuring for co-localization of signals based on overlap does not provide accurate information about whether the proteins are in complex with each other9,10.
Furthermore, co-immunoprecipitation and co-immunostaining of protein-protein complexes are not quantitative, making it challenging to determine if such interactions are significant. FRET and BiFC are both fluorescent-based techniques. FRET relies on tagging proteins of interest with fluorescent proteins (FPs) that have spectral overlap at which energy from one FP (donor) is transferred to another FP (acceptor)11. This nonradiative transfer of energy results in fluorescence of the acceptor FP that can be detected at its respective wavelength of emission. BiFC is based on reconstitution of a fluorescent protein in vivo. It entails splitting GFP into two complementary fragments, such as helices 1-10 and helix 1112, which are then fused to two proteins of interest. If these two proteins interact, the complementary fragments of GFP become close enough in proximity to fold and assemble, reconstituting the GFP fluorophore. Reconstituted GFP is then directly observed as fluorescence and indicates where a PPI has occurred.
As such, both FRET and BiFC depend on large fluorescent tags that can disrupt the function of the tagged protein. In addition, FRET and BiFC require abundant and comparable expression of the tagged proteins to obtain accurate data. FRET may not be suitable for experiments where one partner is in excess of the other, which can lead to high background13. Overexpression in BiFC experiments should also be avoided, as this can induce nonspecific assembly14 that results in increased background. Both techniques require optimization of expression and imaging conditions of the tagged proteins, which may prolong the time required to complete experiments.
The proximity ligation assay (PLA) is an alternative approach that can address the limitations of the techniques mentioned above. PLA takes advantage of primary antibodies that recognize the proteins of interest (or their tags). These primary antibodies are then bound by secondary antibodies containing oligonucleotide probes that can hybridize with one another when within a 40 nm (or shorter) distance15. The resulting hybridized DNA is amplified through a PCR reaction, which is detected by probes that complement the DNA. This results in foci that are visualized by a microscope. This technology can detect PPIs in situ in complex tissues (i.e., the worm gonad), which is organized as an assembly line containing cells at various stages of development and differentiation. With PLA, PPIs can be directly visualized in a fixed worm gonad, which is advantageous for investigating whether PPIs occur during a specific stage of development. PLA offers greater resolution of PPIs as opposed to co-localization-based assays, which is ideal for making precise measurements. If used, super-resolution microscopy has the potential to provide finer detail about the location of PLA foci within a cell. Another advantage is that the foci resulting from PLA reactions can be counted by an ImageJ-based analysis workflow, making this technique quantitative.
The LC8 family of dynein light chains was first described as a subunit of the dynein motor complex16 and hypothesized to serve as a cargo adapter. Since its initial discovery, LC8 has been found in multiple protein complexes in addition to the dynein motor complex17,18,19,20. Scanning for protein sequences that contain the LC8 interaction motif19 suggests that LC8 may have many interactions with a wide array of different proteins17,18,19,20,21,22. As a result, LC8 family proteins are now considered hubs that help promote the assembly of larger protein complexes19,22, such as assemblies of intrinsically disordered proteins21.
One C. elegans LC8-family protein, dynein light chain-1 (DLC-1), is widely expressed across many tissues and not enriched in specific subcellular structures23,24. Consequently, identification of biologically relevant in vivo partners of DLC-1 in C. elegans is challenging for a number of reasons: 1) co-immunoprecipitation does not indicate the tissue source where the interaction occurs; 2) limited expression of particular partners or transient interactions may hinder the ability to detect an interaction by co-immunoprecipitation; and 3) diffuse distribution of DLC-1 leads to non-specific overlap with potential partner proteins by co-immunostaining. Based on these challenges, PLA is an ideal approach for testing in vivo interactions with DLC-1.
It has been previously reported that DLC-1 directly interacts with and serves as a cofactor for the RNA-binding proteins (RBPs) FBF-223 and GLD-125. Our work supports the model of DLC-1 serving as a hub protein and suggests that DLC-1 facilitates an interaction network that spans beyond dynein19,22. Using a GST pulldown assay, a new DLC-1-interacting RBP named OMA-1 has been identified26. OMA-1 is important for oocyte growth and meiotic maturation27 and functions in conjunction with a number of translational repressors and activators28. While FBF-2 and GLD-1 are expressed in the stem cells and meiotic pachytene regions, respectively, OMA-1 is diffusely expressed in the germline from the meiotic pachytene through the oocytes27 (Figure 1). This suggests that DLC-1 forms complexes with RBPs in different regions of the gonad. It has also been found that the direct interaction between DLC-1 and OMA-1 observed in vitro is not recovered by an in vivo IP. The PLA has been successfully used as an alternate approach to further study this interaction in the C. elegans germline, and results suggest that PLA can be used to probe many other PPIs in the worm.

<table><tbody><tr><td>16% paraformaldehyde solution</td><td>Electron Microscopy services</td><td>15710</td><td>used to make 4% working solution</td></tr><tr><td>1M KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td>Prepare a 1M working stock</td></tr><tr><td>1x M9</td><td>various</td><td>various</td><td>prepared as 10x stock used at 1x; see wormbook.org for protocol</td></tr><tr><td>1x PBS</td><td>various</td><td>various</td><td>see wormbook.org for protocol</td></tr><tr><td>26.5 Gauge Needle</td><td>Exel International</td><td>26402</td><td>Needles used for dissection</td></tr><tr><td>BSA</td><td>Lampire</td><td>7500802</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>Thermo Scientific</td><td>05-529C</td><td>50ml Oak ridge centrifuge tube used for synchronization</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>880</td><td></td></tr><tr><td>Coplin Jar</td><td>PolyLab</td><td>62101</td><td></td></tr><tr><td>Coverslip to Freeze Sample</td><td>Globe Scientific</td><td>1411-10</td><td>22x40mm, No. 1</td></tr><tr><td>Coverslip to Seal Slide</td><td>Globe Scientific</td><td>1404-15</td><td>22x22mm, No. 1.5</td></tr><tr><td>DAPI Mounting Medium for Immunofluorescence</td><td>Vector</td><td>H-1200</td><td></td></tr><tr><td>Ligase</td><td>Sigma-Aldrich</td><td>DUO82029</td><td>Duolink 1x Ligase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td></tr><tr><td>Amplification red buffer</td><td>Sigma-Aldrich</td><td>DUO82011</td><td>Duolink 5x Amplification Red buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td></tr><tr><td>Ligation Buffer</td><td>Sigma-Aldrich</td><td>DUO82009</td><td>Duolink 5x Ligation buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td></tr><tr><td>Antibody Diluent</td><td>Sigma-Aldrich</td><td>DUO82008</td><td>Duolink antibody diluent,Comes with DUO92004 and DUO92002, Note: A 1x PBS/1% BSA solution can also be used as a substitute to dilute the antibody.</td></tr><tr><td>Blocking Solution</td><td>Sigma-Aldrich</td><td>DUO82007</td><td>Duolink blocking solution, Comes with DUO92004 and DUO92002</td></tr><tr><td>Mounting Medium for PLA</td><td>Sigma-Aldrich</td><td>DUO82040</td><td>Duolink In Situ mounting medium with DAPI</td></tr><tr><td>MINUS Probe</td><td>Sigma-Aldrich</td><td>DUO92004</td><td>Duolink In Situ Probe Anti-Mouse MINUS</td></tr><tr><td>PLUS Probe</td><td>Sigma-Aldrich</td><td>DUO92002</td><td>Duolink In Situ Probe Anti-Rabbit PLUS</td></tr><tr><td>Wash Buffer A</td><td>Sigma-Aldrich</td><td>DUO82046</td><td>Duolink In Situ wash Buffer A</td></tr><tr><td>Wash Buffer B</td><td>Sigma-Aldrich</td><td>DUO82048</td><td>Duolink In Situ wash Buffer B</td></tr><tr><td>Polymerase</td><td>Sigma-Aldrich</td><td>DUO82030</td><td>Duolink Polymerase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td></tr><tr><td>Epifluorescent Microscope</td><td>Leica</td><td></td><td>DFC300G camera, DM5500B microscope</td></tr><tr><td>Goat anti-mouse Alexa 594</td><td>JacksonImmuno</td><td>115-585-146</td><td>Use at 1:500</td></tr><tr><td>Goat anti-rabbit Alexa 488</td><td>JacksonImmuno</td><td>111-545-144</td><td>Use at 1:200</td></tr><tr><td>Image Processing Software</td><td>Adobe</td><td></td><td>Adobe Photoshop + Illsutrator CS3</td></tr><tr><td>Glass Pipette</td><td>Corning</td><td>7095B-5X</td><td></td></tr><tr><td>Levamisole</td><td>ACROS Organics</td><td>187870100</td><td>Prepare a 250mM working stock</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454</td><td></td></tr><tr><td>Mouse anti-FLAG</td><td>Sigma</td><td>F1804</td><td>Use at 1:1000 for immunofluorescence and PLA, pre-block with normal goat serum recommended</td></tr><tr><td>Nailpolish</td><td>L.A. colors</td><td>CNP195</td><td></td></tr><tr><td>Nematode Growth Medium (NGM)</td><td>various</td><td></td><td>See wormbook.org for protocol</td></tr><tr><td>Normal Goat Serum</td><td>JacksonImmuno</td><td>005-000-121</td><td></td></tr><tr><td>Polyethylene Pasteur Pipette</td><td>Globe Scientific</td><td>135030</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td>Prepared as 0.1% stock solution in water, stored at -20C, and diluted 1:100 in water to coat slides</td></tr><tr><td>Petri Dishes</td><td>Tritech</td><td>PD7060</td><td>60 mm diameter</td></tr><tr><td>Rabbit anti-GFP</td><td>Thermo Fisher</td><td>G10362</td><td>Use at 1:200 for immunofluorescence, 1:4000 for PLA</td></tr><tr><td>Slides</td><td>Thermo Fisher</td><td>30-2066A-Brown</td><td>Three-square 14x14mm autoclavable slides with bars are custom-ordered through Fisher Scientific. Poly-L-Lysine added to slides in the lab</td></tr><tr><td>Sodium Hypochlorite solution</td><td>Fisher Scientific</td><td>SS290-1</td><td></td></tr><tr><td>task wipes</td><td>Kimtech</td><td>34120</td><td>4.4x8.4 inch task wipes</td></tr><tr><td>Trays (242x241x20mm)</td><td>Thermo Fisher</td><td>240845</td><td>Used to make humid chamber</td></tr><tr><td>Triton X-100</td><td>ACROS Organics</td><td>327372500</td><td></td></tr><tr><td>Ultrapure water</td><td>Milli-Q</td><td></td><td>Ultrapure water obtained from Milli-Q Integral Water Purification System</td></tr><tr><td>Watchglass</td><td>Carolina Biological</td><td>742300</td><td></td></tr><tr><td>-20 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>Aluminum Foil</td><td></td><td></td><td></td></tr><tr><td>OP50 strain &lt;em&gt;E. coli&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Orbital Shaker</td><td></td><td></td><td></td></tr><tr><td>Tape</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Nematode strains used in this study (both available upon request)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;mntSi13[pME4.1] II; unc-119(ed3) III; teIs1 [pRL475]&lt;/em&gt;</td><td></td><td>UMT 376</td><td>&lt;em&gt;dlc-1 prom::3xFLAG::dlc-1::dlc-1 3&#039;UTR; oma-1 prom::oma-1::GFP&lt;/em&gt;; Reference 24</td></tr><tr><td>&lt;em&gt;mntSi13[pME4.1] II; mntSi21[pXW6.22] unc-119(ed3) III&lt;/em&gt;</td><td></td><td>UMT 422</td><td>&lt;em&gt;dlc-1 prom::3xFLAG::dlc-1::dlc-1 3&#039;UTR; gld-1 prom::ceGFP::fbf-1 3&#039;UTR + unc-119(+)&lt;/em&gt;; Reference: this study</td></tr></tbody></table>

in situ detection of ribonucleoprotein complex assembly in the c. elegans germline using proximity ligation assay

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.

Co-immunostaining of both 3xFLAG::DLC-1; GFP and 3xFLAG::DLC-1; OMA-1::GFP germlines with FLAG and GFP antibodies revealed their patterns of expression in the germline (Figure 3Aii-iii,3Bii-iii). While GFP was expressed throughout the germline (Figure 3Aiii), OMA-1::GFP expression was restricted to the late pachytene and oocytes (Figure 3Biii)<sup cl...

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 ...

in-situ-detection-ribonucleoprotein-complex-assembly-c-elegans

Research

JoVE Journal

Developmental Biology

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This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

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Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

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Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

We present an immunofluorescence imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the 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.

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

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Learn proximity ligation assay for detecting protein-protein interactions in situ using C. elegans germline. This method reveals spatial and temporal patterns to study developmental regulation.

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

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