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 efficiency, paraformaldehyde solutions should be prepared fresh before every experiment.</li>
	<li>Prepare a humid chamber by covering the bottom of a closed box with wet tissues. Place the humid chamber at 37 &#176;C before starting the experiment, to ensure that the chamber temperature is at 37 &#176;C while incubating the enzymatic reactions.</li>
	<li>Culture HeLa cells in T25 flasks, in 5 mL of DMEM (High Glucose, Glutamate and Sodium Pyruvate supplemented) supplemented with 10% FCS and 1% Pen-Strep, in a 37 &#176;C CO2 incubator containing 5% CO2. Dissociate adherent cells using 0.05% Trypsin-EDTA. After addition of fresh DMEM to dissociated cells, count cells using a cell counting chamber and grow on diagnostic slides inside a humid chamber.</li>
	<li>Sterilize diagnostic slides by UV-irradiation in a sterile cell culture hood for 30 min.</li>
	<li>Add 100 &#181;L of sterile filtered 0.0001% poly-L-lysine to each well required for the experiment. Incubate for 30 min. Wash away excess poly-L-lysine by washing each well 3x with 50 &#181;L ultrapure&#160;water.</li>
	<li>Trypsinize the HeLa cells and add approximately 15,000 cells to each well. If necessary, dilute cells to at least 30-50 &#181;L of DMEM per well.</li>
	<li>Grow cells in a humid chamber within a 37 &#176;C 5% CO2 incubator for approximately 24 h. Cells should be 60%&#8211;80% confluent before commencing the PLA. 
	NOTE: Too high a confluency decreases the absorption of reagents, decreasing the obtained signal at the end of the protocol.</li>
	<li>Remove medium by placing a tissue paper at the edge of the well. Wash wells 3x with 50 &#181;L of PBS. 
	NOTE: Cells are subject to detaching when liquids are added harshly. This can be prevented by not letting the wells dry completely before adding new liquid and by adding the new liquid gently to the edge of the well.</li>
	<li>Fix cells by adding 50 &#181;L of freshly prepared 4% paraformaldehyde in PBS to each well. Incubate for 10 min at room temperature.</li>
	<li>Wash slides 3x in PBS. Perform washes in a Coplin slide-staining jar containing 100 mL of PBS. For each wash, incubate for 5 min at room temperature without shaking.</li>
	<li>Permeabilize the cell membrane by submerging the slides in 100 mL of 0.5% Triton-X100 in PBS in a Coplin slide-staining jar. Incubate for 10 min at room temperature without shaking.</li>
	<li>Wash slides 3x in TBS-T. Perform washes in a Coplin slide-staining jar containing 100 mL of TBS-T. For each wash, incubate for 5 min at room temperature without shaking.</li>
	<li>After the last wash, remove excess buffer with a tissue paper. At this point the cells are ready for the Proximity Ligation Assay protocol that will be discussed in section 4.</li>
</ol>
2. S. cerevisiae Cell Preparation
<ol>
	<li>Prepare the following materials: 100 mM KPO4, pH 6.5, referred to as Wash Buffer; 37% formaldehyde; 4% paraformaldehyde in 100 mM KPO4, pH 6.5; 1.2 M sorbitol in 100 mM KPO4, pH 6.5; lyticase solution (500 &#181;g/mL lyticase, 20 mM &#946;-mercaptoethanol, 100 mM KPO4, pH 6.5); 0.0001% poly-L-Lysine solution; 1% Triton-X100 in 100 mM KPO4, pH 6.5; 10-well diagnostic slides; a humid chamber (prepared as in step 1.2); tissue paper; and Coplin slide-staining jars. 
	NOTE: To ensure optimal fixation efficiency, paraformaldehyde solutions should be prepared fresh before every experiment.</li>
	<li>Grow an overnight culture in non-selective Yeast Extract, Peptone and Dextrose (YPD) medium at 30 &#176;C while shaking. 
	NOTE: Depending on experimental requirements S. cerevisiae cells can be grown in Synthetic Complete (SC) medium or selective Synthetic Minimal (SM) medium instead.</li>
	<li>Dilute the stationary culture to an OD600 of 0.1 in 20 mL medium. Grow the cells at 30 &#176;C while shaking until the OD600 reaches 0.5.</li>
	<li>Transfer the 20 mL culture to a 50 mL centrifuge tube. Pellet the cells by centrifugation at 665 x g for 3 min. Remove the supernatant. Resuspend the cells in 5 mL of fresh medium.</li>
	<li>Fix the cells by adding 550 &#181;L of 37% formaldehyde to the culture. Incubate at room temperature for 15 min.</li>
	<li>Pellet the cells by centrifugation at 665 x g for 3 min. Remove the supernatant. Resuspend the pellet in 1 mL of freshly prepared 4% paraformaldehyde in Wash Buffer. Incubate for 45 min at room temperature.</li>
	<li>During the incubation, prepare the diagnostic slides by adding 100 &#181;L of 0.01% poly-L-lysine solution to each well. Incubate the slides for 30 min at room temperature.</li>
	<li>After 30 min, wash away excess poly-L-lysine with ultrapure water and let the slides air dry. The dry slides are ready for use.</li>
	<li>Wash cells twice with 1 mL of Wash Buffer. Perform washes by centrifugation of the cells at 665 x g for 3 min. Remove the supernatant and resuspend the cells in Wash Buffer.</li>
	<li>Pellet the cells by centrifugation at 665 x g for 3 min. Remove the supernatant. Resuspend the cells in 1 mL of 1.2 M sorbitol in Wash Buffer.</li>
	<li>Pellet the cells by centrifugation at 665 x g for 3 min. Remove the supernatant. Resuspend the pellet in 250 &#181;L of freshly prepared Lyticase solution to digest the cell wall. Incubate the cells in Lyticase solution for 15 min at 30 &#176;C while shaking.</li>
	<li>After digestion, wash cells 3x by centrifugation at 665 x g for 3 min and remove the supernatant. Resuspend the cells in 250 &#181;L of 1.2 M sorbitol in Wash Buffer. 
	NOTE: Because cells are fragile after cell wall digestion, resuspend them very carefully to not damage the cells.</li>
	<li>Add 20 &#181;L of resuspended cells to the poly-L-lysine coated slides. Allow them to attach to the slides for 30 min. Wash away non-adherent cells by washing the wells 3x with 50 &#181;L of Wash Buffer.</li>
	<li>Permeabilize the cell membrane by washing 3x with 50 &#181;L of 1% Triton-X in Wash Buffer. 
	NOTE: At this point the cells are ready for the proximity ligation assay protocol that will be discussed in section 4.</li>
</ol>
3. E. coli Cell Preparation
<ol>
	<li>Prepare the following materials: PBS-T (140 mM NaCl, 2 mM KCl, 8 mM K2HPO4, 1.5 mM KH2PO4, 0.05% Tween-20), pH 7.4; lysozyme solution (2 mg/mL lysozyme, 25 mM Tris-HCl pH 8.0, 50 mM Glucose, 10 mM EDTA); 0.0001% poly-L-Lysine solution; 99% ice-cold methanol; 99% room temperature methanol; 99% acetone; 10-well diagnostic slides; a humid chamber (prepared as in step 1.1.1); tissue paper; and Coplin slide-staining jars.</li>
	<li>Grow an overnight culture in Luria-Bertani (LB) medium at 30 &#176;C while shaking.</li>
	<li>Dilute the stationary culture to an OD600 of 0.02 in fresh LB medium. Grow the cells at 30 &#176;C while shaking until the OD600 reaches 0.4 for log phase cells.</li>
	<li>Approximately 15 min before cells reach an OD600 of 0.4, prepare poly-L-lysine coated slides by adding 100 &#181;L of 0.0001% poly-L-lysine to each well. Incubate the slides for 30 min at room temperature.</li>
	<li>After 30 min wash away excess poly-L-lysine with ultrapure water and let slides air dry. The dry slides are ready for use.</li>
	<li>When the cells reach an OD600 of 0.4, transfer 1 mL of the culture to a sterile microcentrifuge tube and pellet cells at 2,650 x g for 2 min.</li>
	<li>Resuspend cells in 50 &#181;L of LB medium.</li>
	<li>Fix cells by adding 1 mL of ice-cold 99% methanol. Mix very gently by hand. Incubate cells for 30 min at -20 &#176;C.</li>
	<li>After fixation add 20 &#181;L of the cells to the poly-L-lysine coated slides. Let the slides air dry for 30 min.</li>
	<li>Add 50 &#181;L of freshly prepared lysozyme solution to each well to digest the cell wall. Incubate in a humid chamber for 30 min at 25 &#176;C.</li>
	<li>Remove the lysozyme solution from the wells by adding a tissue paper to the edge of the well. Wash slides 3x in 100 mL of PBS-T. Perform each wash in a Coplin slide-staining jar for 30 s, without shaking.</li>
	<li>Remove wash buffer from the slides by tapping the slides on a tissue paper.</li>
	<li>Permeabilize the cell membranes by adding 50 &#181;L of 99% methanol to each well. Incubate for 1 min at room temperature.</li>
	<li>Remove methanol by placing a tissue paper to the edge of the well.</li>
	<li>Add 50 &#181;L of 99% acetone to each well. Incubate for 1 min.</li>
	<li>Remove excess acetone by placing a tissue paper to the edge of the well. Allow slides to air dry. At this point the cells are ready for the proximity ligation assay protocol that will be discussed in section 4.</li>
</ol>
4. Proximity Ligation Assay
<ol>
	<li>Prepare the following materials: Blocking Buffer; Antibody Dilution Buffer; Wash Buffer &#8216;A&#8217; &#8211; (10 mM Tris, 150 mM NaCl, 0.05% Tween-20) pH 7.4; Wash Buffer &#8216;B&#8217; &#8211; (200 mM Tris, 100 mM NaCl) pH 7.4; Anti-Rabbit Secondary Antibody PLUS; Anti-Mouse Secondary Antibody MINUS; 5x Ligation Buffer; ligase; 5x Amplification Buffer (Orange: &#955;ex&#160;554&#160;nm; &#955;em&#160;576&#160;nm); polymerase; ultrapure water; and mounting medium containing DAPI. 
	NOTE: The PLA detection reagents are also available in the variants Green (&#955;ex 495 nm; &#955;em 527 nm), Red (&#955;ex 594 nm; &#955;em 624 nm), FarRed (&#955;ex 644 nm; &#955;em 669 nm) or Brightfield (horseradish peroxidase (HRP) conjugated).</li>
	<li>Block the cells by adding a drop of Blocking Buffer to each well. Incubate for 30 min at 37 &#176;C in a humid chamber.</li>
	<li>Prepare antibody solutions by diluting stock antibodies in Antibody Dilution Buffer. For each well 40 &#181;L of antibody solution is required.</li>
	<li>Remove blocking buffer by placing a tissue paper at the edge of the well. Add 40 &#181;L of antibody diluted in Antibody Dilution Buffer to each well. Incubate for 60 min in a humid chamber at 37 &#176;C or overnight at 4 &#176;C.</li>
	<li>Remove antibody solution from the wells by placing a tissue paper at the edge of the well. Wash slides 2x in 100 mL of Wash Buffer &#8216;A&#8217; in a Coplin slide-staining jar for 5 min, without shaking.</li>
	<li>During the wash steps, dilute 5x secondary antibody probes, anti-rabbit PLUS &#38; anti-mouse MINUS (the species specificity of the probes depends on the primary antibodies used), in Antibody Dilution Buffer. Prepare 40 &#181;L of antibody solution per well.</li>
	<li>Add 40 &#181;L of secondary antibody solution to each well. Incubate for 60 min at 37 &#176;C in a humid chamber.</li>
	<li>Remove secondary antibody solution from the wells by placing a tissue paper at the edge of the well. Wash slides 2x in 100 mL of Wash Buffer &#8216;A&#8217; in a Coplin slide-staining jar for 5 min, without shaking.</li>
	<li>During the washes prepare 40 &#181;L of ligation mixture per well, by mixing 8 &#181;L of 5x Ligation Buffer, 31 &#181;L of ultrapure water and 1 &#181;L of ligase.</li>
	<li>Add 40 &#181;L of ligation mixture to each well. Incubate for 30 min at 37 &#176;C in a humid chamber.</li>
	<li>Remove ligation mixture from the wells by placing a tissue paper at the edge of the well. Wash slides 2x in 100 mL of Wash Buffer &#8216;A&#8217; in a Coplin slide-staining jar for 2 min, without shaking.</li>
	<li>During the washes prepare 40 &#181;L of amplification mixture per well, by mixing 8 &#181;L of 5x amplification solution, 31.5 &#181;L of ultrapure water, and 0.5 &#181;L of polymerase. 
	NOTE: The 5x amplification contains fluorescent probes. Protect this mixture from light. Also, protect the slides from light during each of the following steps. If using translucent Coplin slide-staining jars and humid chambers, wrap them in aluminum foil.</li>
	<li>Add 40 &#181;L of amplification mixture per well. Incubate for 100 min at 37 &#176;C in a humid chamber.</li>
	<li>Remove amplification mixture from the wells. Wash slides 2x in 100 mL of Wash Buffer &#8216;B&#8217; in a Coplin slide-staining jar for 10 min without shaking.</li>
	<li>Wash slides in 100 mL of Wash Buffer &#8216;B&#8217; diluted 1:100 in ultrapure water in a Coplin slide-staining jar for 30 s.</li>
	<li>Add 20 &#181;L of DAPI containing mounting medium per well to the slides. Close slides with a coverslip and seal slides with nail polish.</li>
	<li>If imaging, immediately incubate DAPI containing mounting medium for 10&#8211;15 min, while protected from light. If not, store the slides at -20 &#176;C for up to 1 week, protected from light.</li>
</ol>
5. Detection
<ol>
	<li>Use confocal microscopy to acquire images of HeLa, S. cerevisiae and E. coli cells with 20x/0.8 NA, 63x/1.4 NA and 100x/1.4 NA Plan Apochromat objectives, respectively. Excite DNA-stained DAPI with a 405 nm pulsed diode laser. For the PLA signal (for this study) excite with a 561 nm solid-state laser.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+stationary+phase+induced+by+low+oxygen+tension:+cell+wall+thickening+and+localization+of+the+16-kilodalton+alpha-crystallin+homolog.">Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog.</a> Journal of Bacteriology. 180 (4), 801-808 (1998).</li><li>Werner-Washburne, M., Braun, E., Johnston, G. C., Singer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stationary+phase+in+the+yeast+Saccharomyces+cerevisiae.">Stationary phase in the yeast Saccharomyces cerevisiae.</a> Microbiological Reviews. 57 (2), 383-401 (1993).</li></ol>

The authors have nothing to disclose.

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...

Watch this Scientific Journal Video about In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells at JoVE.com

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 ...

in-situ-monitoring-transiently-formed-molecular-chaperone-assemblies

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JoVE Journal

Biology

7.0K Views.  University of Groningen. 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.

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

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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

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