In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Summary

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Abstract

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Introduction

A vast amount of genomic information remains uninterpretable due to our incomplete understanding of cellular interactomes. Conventional protein-protein interaction detection methodologies such as protein co-immunoprecipitation with/without chemical cross-linking and protein co-localization, though widely used, pose a range of disadvantages. Some  of the main disadvantages include poor quantification of the interactions and the potential introduction of non-native binding events. In comparison, emerging proximity-based techniques provide an alternative and a powerful approach for capturing protein interactions in cells. The proximity ligation assay (PLA)

Protocol

1. HeLa Cell Preparation

1. Prepare the following materials: PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), pH 7.4; DMEM, supplemented with 10% FCS and 1% Pen-Strep; 4% paraformaldehyde in PBS; 0.5% Triton-X100 in PBS; TBS-T (150 mM NaCl, 20 mM Tris, 0.05% Tween), pH 7.4; 0.0001% sterile poly-L-Lysine solution; 10-well diagnostic slides; a humid chamber; tissue paper; and Coplin slide-staining jars.
   NOTE: To ensure optimal fixation efficiency, paraformaldehyde solutions should be prepared fresh before every experiment.
2. Prepare a humid chamber by covering the bottom of ....

Results

Our previous in vitro studies using purified proteins revealed that a subset of human class A and class B JDPs form transient mixed class JDP complexes to efficiently target a broad range of aggregated proteins and possibly facilitate the assembly of Hsp70-based protein disaggregases⁷. We employed PLA to determine whether mixed class (A+B) JDP complexes occur in human cervical cancer cells (HeLa). Human JDPs DNAJA2 (class A) and DNAJB1 (class B) were targeted with highly specific primary anti.......

Discussion

Co-immunoprecipitation and co-localization based approaches have been used as long-standing methods to characterize protein assembles. The detection of transiently formed specific chaperone complexes is a major challenge with such conventional methods, and as a result, previous findings are largely restricted to qualitative  interpretations. The cell lysis-based co-immunoprecipitation techniques often require cross-linking to stabilize protein-protein interactions. Cell lysis increases the risk of disrupting tr.......

Materials

Name	Company	Catalog Number	Comments
37% Formaldehyde	Merck	103999
Acetone	Sigma-Aldrich	32201
anti-DNAJA2 antibody	Abcam	ab157216
anti-DNAJB1 Antibody	Enzo Life Sciences	ADI-SPA-450
anti-DnaK antibody	In house
anti-mCherry antibody	Abcam	ab125096
anti-Sis1 Antibody	Cosmo Bio Corp	COP-080051
anti-Ydj1 antibody	StressMarq Biosciences	SMC-150,
anti-YFP antibody	In house
Coplin slide-staining jar	Sigma-Aldrich	S5516
Diagnostic slides	Marienfeld	1216530
DMEM	Thermo-Fischer	31966021
DuoLink In Situ Detection Reagents Orange	Sigma-Aldrich	DUO92007
DuoLink In Situ Mounting Medium + DAPI	Sigma-Aldrich	DUO82040
DuoLink In Situ PLA Probe Anti-Mouse MINUS	Sigma-Aldrich	DUO92004
DuoLink In Situ PLA Probe Anti-Rabbit PLUS	Sigma-Aldrich	DUO92002
DuoLink In Situ Wash Buffers, Fluorescence	Sigma-Aldrich	DUO82049
Fetal Calf Serum	Thermo-Fischer	10082147
Lysozyme	Sigma-Aldrich	62971
Methanol	Sigma-Aldrich	32213
Paraformaldehyde	Sigma-Aldrich	P6148
Penicillin/Streptomycin	Thermo-Fischer	15070063
Poly-L-Lysine	Sigma-Aldrich	P47-07
Sorbitol	Sigma-Aldrich	S7547
Triton-X100	Merck	108643
Trypsin	Thermo-Fischer	25300096
Tween-20	Sigma-Aldrich	P1379
Zymolase 100T / / Lyticase	United States Biological	Z1004