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

Summary

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

Abstract

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.

Introduction

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, ....

Protocol

NOTE: This protocol uses C. elegans strains in which potential interacting partners are both tagged. It is strongly recommended that a negative control strain be used, in which one tagged protein is not expected to interact with another tagged candidate interaction partner. Here, GFP alone was used as a negative control to assess background, as DLC-1 is not expected to interact with GFP in the worm. GFP-tagged OMA-1 was used as the experimental strain, as preliminary data suggest an interaction with DLC-1. Nematode strains co-expressing control and test proteins with 3xFLAG-tagged DLC-1 are referred to in this text as 3xFLAG::DLC-1; GFP and 3xFLAG::DLC-1; OMA....

Results

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)

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% paraformaldehyde solution	Electron Microscopy services	15710	used to make 4% working solution
1M KH2PO4	Sigma	P0662	Prepare a 1M working stock
1x M9	various	various	prepared as 10x stock used at 1x; see wormbook.org for protocol
1x PBS	various	various	see wormbook.org for protocol
26.5 Gauge Needle	Exel International	26402	Needles used for dissection
BSA	Lampire	7500802
Centrifuge Tubes	Thermo Scientific	05-529C	50ml Oak ridge centrifuge tube used for synchronization
Confocal Microscope	Zeiss	880
Coplin Jar	PolyLab	62101
Coverslip to Freeze Sample	Globe Scientific	1411-10	22x40mm, No. 1
Coverslip to Seal Slide	Globe Scientific	1404-15	22x22mm, No. 1.5
DAPI Mounting Medium for Immunofluorescence	Vector	H-1200
Ligase	Sigma-Aldrich	DUO82029	Duolink 1x Ligase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008
Amplification red buffer	Sigma-Aldrich	DUO82011	Duolink 5x Amplification Red buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008
Ligation Buffer	Sigma-Aldrich	DUO82009	Duolink 5x Ligation buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008
Antibody Diluent	Sigma-Aldrich	DUO82008	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.
Blocking Solution	Sigma-Aldrich	DUO82007	Duolink blocking solution, Comes with DUO92004 and DUO92002
Mounting Medium for PLA	Sigma-Aldrich	DUO82040	Duolink In Situ mounting medium with DAPI
MINUS Probe	Sigma-Aldrich	DUO92004	Duolink In Situ Probe Anti-Mouse MINUS
PLUS Probe	Sigma-Aldrich	DUO92002	Duolink In Situ Probe Anti-Rabbit PLUS
Wash Buffer A	Sigma-Aldrich	DUO82046	Duolink In Situ wash Buffer A
Wash Buffer B	Sigma-Aldrich	DUO82048	Duolink In Situ wash Buffer B
Polymerase	Sigma-Aldrich	DUO82030	Duolink Polymerase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008
Epifluorescent Microscope	Leica		DFC300G camera, DM5500B microscope
Goat anti-mouse Alexa 594	JacksonImmuno	115-585-146	Use at 1:500
Goat anti-rabbit Alexa 488	JacksonImmuno	111-545-144	Use at 1:200
Image Processing Software	Adobe		Adobe Photoshop + Illsutrator CS3
Glass Pipette	Corning	7095B-5X
Levamisole	ACROS Organics	187870100	Prepare a 250mM working stock
Methanol	Fisher Scientific	A454
Mouse anti-FLAG	Sigma	F1804	Use at 1:1000 for immunofluorescence and PLA, pre-block with normal goat serum recommended
Nailpolish	L.A. colors	CNP195
Nematode Growth Medium (NGM)	various		See wormbook.org for protocol
Normal Goat Serum	JacksonImmuno	005-000-121
Polyethylene Pasteur Pipette	Globe Scientific	135030
Poly-L-Lysine	Sigma-Aldrich	P1524	Prepared as 0.1% stock solution in water, stored at -20C, and diluted 1:100 in water to coat slides
Petri Dishes	Tritech	PD7060	60 mm diameter
Rabbit anti-GFP	Thermo Fisher	G10362	Use at 1:200 for immunofluorescence, 1:4000 for PLA
Slides	Thermo Fisher	30-2066A-Brown	Three-square 14x14mm autoclavable slides with bars are custom-ordered through Fisher Scientific. Poly-L-Lysine added to slides in the lab
Sodium Hypochlorite solution	Fisher Scientific	SS290-1
task wipes	Kimtech	34120	4.4x8.4 inch task wipes
Trays (242x241x20mm)	Thermo Fisher	240845	Used to make humid chamber
Triton X-100	ACROS Organics	327372500
Ultrapure water	Milli-Q		Ultrapure water obtained from Milli-Q Integral Water Purification System
Watchglass	Carolina Biological	742300
-20 °C freezer
-80 °C freezer
Aluminum Foil
OP50 strain E. coli
Orbital Shaker
Tape
Nematode strains used in this study (both available upon request)
mntSi13[pME4.1] II; unc-119(ed3) III; teIs1 [pRL475]		UMT 376	dlc-1 prom::3xFLAG::dlc-1::dlc-1 3'UTR; oma-1 prom::oma-1::GFP; Reference 24
mntSi13[pME4.1] II; mntSi21[pXW6.22] unc-119(ed3) III		UMT 422	dlc-1 prom::3xFLAG::dlc-1::dlc-1 3'UTR; gld-1 prom::ceGFP::fbf-1 3'UTR + unc-119(+); Reference: this study