Name: in situ monitoring of transiently formed molecular chaperone assemblies in bacteria, yeast, and human cells
Uploaded: 2019-09-02T15:00:00
Description: Watch this Scientific Journal Video about In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells at JoVE.com

NBN is supported by a special Recruitment Grant from the Monash University Faculty of Medicine Nursing and Health Sciences with funding from the State Government of Victoria and the Australian Government. We thank Bernd Bukau (ZMBH, Heidelberg University, Germany) and Harm H. Kampinga (Department of Biomedical Sciences of Cells &#38; Systems, University of Groningen, The Netherlands) for their invaluable support and sharing of reagents, Holger Lorenz (ZMBH Imaging Facility, Heidelberg University, Germany) for his support with confocal microscopy and image processing, and Claire Hirst (ARMI, Monash University, Australia) for critical reading of the manuscript.

1. HeLa Cell Preparation
<ol>
	<li>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...

<ol>
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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&#160; interpretations. The cell lysis-based co-immunoprecipitation techniques often require cross-linking to stabilize protein-protein interactions.&#160;Cell lysis increases the risk of disrupting tr...

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&#160; of the main disadvantages include poor quantification of the interactions and the potential introduction of non-native binding events.&#160;In comparison, emerging proximity-based techniques provide an alternative and a powerful approach for capturing protein interactions in cells. The proximity ligation assay (PLA)1, now available as a proprietary kit, employs&#160;antibodies to specifically target protein complexes based on the proximity of the interacting subunits.
PLA is initiated by the&#160; formation of a scaffold consisting of primary and secondary antibodies with small DNA tags (PLA probes) on the surface of the targeted protein complex (Figure 1, steps 1-3). Next, determined by the proximity of the DNA tags, a circular DNA molecule is generated via hybridization with connector oligonucleotides (Figure 1, step 4). The formation of the circular DNA is completed by a DNA ligation step. The newly formed circular piece of DNA serves as a template for the subsequent&#160;rolling circle amplification (RCA)-based polymerase chain reaction (PCR) primed by one of the conjugated oligonucleotide tags. This generates a single-stranded concatemeric DNA-molecule attached to the protein complex via the antibody scaffold (Figure 1, step 6). The concatemeric DNA molecule is visualized using fluorescently labeled oligonucleotides that hybridize to multiple unique sequences scattered across the amplified DNA&#160;(Figure 1, step 7)2. The generated PLA signal, which appears as a fluorescent dot (Figure 1, step 7), corresponds to the location of the targeted protein complex in the cell. As a result, the assay could detect protein complexes with high spatial accuracy. The technique is not limited to simply capturing protein interactions, but could also be utilized to detect single molecules or protein modifications on proteins with high sensitivity1,2.
Hsp70 forms a highly versatile chaperone system fundamentally important for maintaining cellular protein homeostasis by participating in an array of&#160;housekeeping and stress-associated functions. Housekeeping activities of the Hsp70 chaperone system include&#160;de novo protein folding, protein translocation across cellular membranes, assembly and disassembly of protein complexes, regulation of protein activity and linking different protein folding/quality control machineries3. The same chaperone system also refolds misfolded/unfolded proteins, prevents protein aggregation, promotes protein disaggregation and cooperates with cellular proteases to degrade terminally misfolded/damaged proteins to facilitate cellular repair after proteotoxic stresses4,5. To achieve this functional diversity, the Hsp70 chaperone relies on partnering co-chaperones of the JDP family and nucleotide exchange factors (NEFs) that fine-tune the Hsp70&#8217;s ATP-dependent allosteric control of substrate binding and release3,6. Further, the JDP co-chaperones play a vital role in&#160;selecting&#160;substrates for&#160;this versatile chaperone system.&#160;Members of this family are subdivided into three classes (A, B and C) based on their structural homology to the prototype JDP, the E. coli DnaJ. Class A JDPs contain an N-terminal J-domain, which interacts with Hsp70, a glycine-phenylalanine rich region, a substrate binding region consisting of a Zinc finger-like region (ZFLR) and two &#946;-barrel domains, and a C-terminal dimerization domain. JDPs with an N-terminal J-domain and a glycine-phenylalanine rich region, but lacking the ZFLR, fall into class B. In general, members of these two classes are&#160;involved in chaperoning functions. Members falling under the catchall class C, which contains JDPs that only share the J-domain4, recruit Hsp70s to perform a variety of non-chaperoning functions. The important role of JDPs as interchangeable substrate recognition &#8220;adaptors&#8221; of the Hsp70 system is reflected by an expansion of the family members during evolution. For example, humans have over 42 distinct JDP members4. These JDPs function as monomers, homodimers and/or homo/hetero oligomers4,5. Recently, a functional cooperation via transient complex formation between class A (e.g., H. sapiens&#160;DNAJA2; S. cerevisiae Ydj1) and class B (e.g., H. sapiens DNAJB1; S. cerevisiae Sis1) eukaryotic JDPs was reported to promote efficient recognition of amorphous protein aggregates in vitro7,8. These mixed class JDP complexes presumably assemble on the surface of aggregated proteins to facilitate the formation of Hsp70- and Hsp70+Hsp100-based protein disaggregases7,8,9,10. The critical evidence to support the existence of these transiently formed mixed class JDP complexes in eukaryotic cells was provided with PLA8.
PLA is increasingly employed for assessing protein interactions in metazoa, primarily in mammalian cells. Here, we report the successful expansion of this&#160;technique to monitor transiently formed chaperone complexes in eukaryotic and prokaryotic unicellular organisms such as the budding yeast S. cerevisiae and the bacterium E. coli.&#160;Importantly, this expansion highlights the potential use of PLA in detecting and analyzing microbes that infect human and animal cells.

<table>
	<tbody>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Merck</td>
			<td>103999</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DNAJA2 antibody</td>
			<td>Abcam</td>
			<td>ab157216</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DNAJB1 Antibody</td>
			<td>Enzo Life Sciences</td>
			<td>ADI-SPA-450</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DnaK antibody</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mCherry antibody</td>
			<td>Abcam</td>
			<td>ab125096</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sis1 Antibody</td>
			<td>Cosmo Bio Corp</td>
			<td>COP-080051</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Ydj1 antibody</td>
			<td>StressMarq Biosciences</td>
			<td>&#160;SMC-150,</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-YFP antibody</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin slide-staining jar</td>
			<td>Sigma-Aldrich</td>
			<td>S5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Diagnostic slides</td>
			<td>Marienfeld</td>
			<td>1216530</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo-Fischer</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Detection Reagents Orange</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92007</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Mounting Medium + DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ PLA Probe Anti-Mouse MINUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Wash Buffers, Fluorescence</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Thermo-Fischer</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>62971</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fischer</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P47-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S7547</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Merck</td>
			<td>108643</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo-Fischer</td>
			<td>25300096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolase 100T / / Lyticase</td>
			<td>United States Biological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ monitoring of transiently formed molecular chaperone assemblies in bacteria, yeast, and human cells

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&#160;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.

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&#160;human cervical cancer cells (HeLa). Human JDPs DNAJA2 (class A) and DNAJB1 (class B) were targeted with highly specific primary anti...

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

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.

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 ...

This method captures transient protein interactions in organisms like bacteria and yeast and in different cell types. Here we monitor transiently formed specific chaperone complexes that are involved in cellular proteostasis. The main advantage of this technique is the ability to generate information on dynamics and sub-cellular localization of protein assemblies in prokaryotic and eukaryotic cells.Adaptations of this technique in unicellular organisms like bacteria and yeast, allows researchers to use it as a powerful tool to investigate diseases related to microbial infections. This technique could potentially be employed to analyze protein interactions in almost all organisms by simply changing the conditions at the sample preparation phase of the protocol. It is important to select good quality antibodies that are specific and tested in either immunohistochemistry, immunofluorescence, ELISA, or immunoprecipitations.Begin by pre-treating 10-well diagnostic slides with poly-L-Lysine. Add 100 microliters of sterile and filtered 0.0001%poly-L-Lysine to each well, and incubate the slides for 30 minutes in a sterile laminar flow hood. Then, wash each well with 50 microliters of sterile water to remove excess poly-L-Lysine.Add approximately 15, 000 cells per well depending on the cell type, and grow the cells in a sterile humid incubator at 37 degrees Celsius with 5%carbon dioxide to a 60 to 80%confluency. After 24 hours, remove the growth medium by placing a tissue paper at the edge of each well, and wash the cells three times with 50 microliters of PBS. Then fix the cells by adding 50 microliters of freshly prepared 4%paraformaldehyde in PBS to each well and incubating the slide at room temperature for 10 minutes.After fixation, wash the slide three times by submerging the slide in a slide-staining jar with PBS. To permeabilize the membranes of the attached cells, submerge the slide in 100 milliliters of 0.5%Triton-X100 in PBS, and incubate the cells for 10 minutes at room temperature. Then wash the slides three times in TBS-T.After the last wash, remove excess buffer with a tissue paper. Grow and dilute S.cerevisiae cells according to manuscript directions, then transfer them to a 50-milliliter centrifuge tube and pellet by centrifugation at 665 times g for three minutes. Remove the supernatant and resuspend the cells in five milliliters of fresh medium.Then add 550 microliters of 37%formaldehyde for a final concentration of 4%and incubate the culture at room temperature for 15 minutes to fix the cells. After the incubation, pellet the cells, remove the supernatant, and resuspend the pellet in one milliliter of freshly prepared 4%paraformaldehyde in wash buffer. Incubate the cells at room temperature for 45 minutes.Meanwhile, prepare the diagnostic slides by adding 100 microliters of 0.0001%poly-L-Lysine solution to each well and incubating the slides for 30 minutes at room temperature. Then wash away the excess poly-L-Lysine with ultrapure water and let the slides air-dry. After the incubation step, wash the cells twice with one milliliter of wash buffer by centrifugation.Remove the supernatant and resuspend the cells in wash buffer. Pellet the cells after the last wash, and remove the supernatant and resuspend the cells in one milliliter of wash buffer containing 1.2 molar sorbitol. To digest the cell wall, pellet the cells again and resuspend the cells in 250 microliters of freshly prepared Lyticase solution.Incubate the cells at 30 degrees Celsius for 15 minutes with gentle shaking. After the digestion step, wash the cells three times in wash buffer and resuspend the cells in 250 microliters of wash buffer containing 1.2 molar sorbitol. Add 20 microliters of re-suspended cells to each poly-L-Lysine-coated well, and allow the fixed and permeabilized cells to attach for 30 minutes.Wash the wells three times with 50 microliters of wash buffer to remove non-adherent cells, and proceed with the proximity ligation assay. Initiate the proximity ligation assay, or PLA protocol, by adding a drop of the blocking buffer to each well and incubate the slide in a humid chamber for 30 minutes at 37 degrees Celsius. Remove the blocking buffer by placing a tissue paper at the edge of each well, and wash the cells twice in 100 milliliters of wash buffer A.Next, add 40 microliters of the primary antibody solution to each well, and incubate the slide for 60 minutes at 37 degrees Celsius in a humid chamber.Remove the primary antibody solution and wash the slides twice in wash buffer A.After the wash, add 40 microliters of the secondary antibody solution to each well and incubate the slide for 60 minutes at 37 degrees Celsius in a humid chamber. Then remove the solution and wash the slides twice in wash buffer A.Then add 40 microliters of the DNA ligation mixture which contains the connector oligonucleotides and DNA ligase, to each well, and incubate the slide in the humid chamber at 37 degrees Celsius for 30 minutes. After incubation, remove the ligation mixture and wash the slides twice with wash buffer A.Add 40 microliters of DNA amplification mixture which contains the DNA polymerase and the fluorescently labeled oligonucleotides to each well, and incubate for 100 minutes in the humid chamber at 37 degrees Celsius.Remove the amplification mixture and wash the slides in 100 milliliters of wash buffer B for 10 minutes per wash. Then wash the slides in 100 milliliters of wash buffer B diluted one to 100 in ultrapure water. Add 20 microliters of DAPI-containing mounting medium per well.Cover the wells of the slide with a cover slip and seal with nail polish. This protocol was successfully used to monitor transient chaperone assemblies formed between distinct J-domain proteins and Hsp70 chaperone and J-domain proteins in HeLa, S.cerevisiae, and E.coli cells. Human class A and class B J-domain proteins were targeted with highly specific primary antibodies.Each of the red fluorescent puncti represents a protein complex formed between two distinct J-domain proteins in HeLa cells. Similar mixed-class J-domain protein complexes are also observed in the unicellular eukaryote S.cerevisiae, or baker's yeast. Some of the fluorescent puncti generated from PLA were more difficult to resolve as individual foci, due to the small cell size.Complex formation between class A and B J-domain proteins were not observed in E.coli cells, which is consistent with biochemical studies performed with purified proteins. But other chaperone assemblies involving J-domain proteins and the Hsp70 chaperone were captured using the PLA protocol. Technical controls lacking a primary antibody against one of the interacting J-domain proteins or chaperones in the otherwise complete PLA reactions showed little or no fluorescence puncti formation in the cells, indicating a lack of false positive signal amplification.To obtain reliable results using the proximity ligation assay, verify beforehand that the antibodies used are of sufficient quality. Furthermore, take precaution in avoiding over-digestion of cells containing a cell wall.

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