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

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...

Watch this Scientific Journal Video about In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay at JoVE.com

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.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

5.8K Views.  University of Montana. This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

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

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

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

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.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

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 ...

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

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

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using 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.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neuroscience

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

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).

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

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

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

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

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.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Source: Wang, X., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60982">In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.</a>&#160;J. Vis. Exp.&#160; (2020)
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.

Test ligacji zbliżeniowej do badania interakcji białko-białko in situ

Source: Wang, X., et al., In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.&#160;J. Vis. ...

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

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Rozdziały w tym wideo

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

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

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

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

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

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

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Test ligacji zbliżeniowej do badania interakcji białko-białko in situ