Name: study of protein-protein interactions in autophagy research
Uploaded: 2017-09-09T13:00:00.000+00:00
Duration: 14 min 8 s
Description: Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

This work was supported by Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant 107T153, the Sabanci University. SEB and NMK are supported by a TUBITAK BIDEB 2211 Scholarship for Ph.D. studies.

1. Immunofluorescence
<ol>
	<li>Maintain HEK293T human embryonic kidney cells in DMEM high glucose medium, and N2A mouse neuroblast cells in DMEM low glucose medium, in a 5% CO2-humidified incubator at 37 &#176;C. Supplement the culture media with 10% heat-inactivated fetal bovine (FBS) serum, antibiotics (50 U/mL penicillin, 50 &#181;g/mL streptomycin), and L-glutamine (2 mM).</li>
	<li>Detach the cells by using 0.25% trypsin. First remove the media of the cell culture then wash the cells with 10 mL sterile phosphate-buffered saline (PBS) and incubate with 1 mL of trypsin at 37 &#176;C for 5 min. Inactivate trypsin by adding 2 mL DMEM medium.</li>
	<li>Wash the plate well by pipetting. Remove 10 &#181;L of cell solution and mix it with 10 &#181;L of trypan blue. Count the number of live cells using a hemocytometer.</li>
	<li>Both HEK293T and N2A are adherent cell lines, however, HEK293T cells detach from the plate easily. Therefore, if working with HEK293T cell line, coat the coverslides with sterile filtered 0.01% poly-L-lysine before seeding the cells.
	<ol>
		<li>Place sterile coverslides in a 10 cm Petri dish. Add the appropriate amount of poly-L-lysine solution onto each coverslide. Incubate the coverslides with poly-L-lysine solution for 10 min.</li>
		<li>Remove the poly-L-lysine solution. 
		Note: Poly-L-lysine is re-usable; it is optional to collect and reuse it for further experiments. Since poly-L-lysine is toxic, wait until all of the solution on the coverslides completely evaporates, and then recover the poly-L-lysine.</li>
		<li>Wash the coverslides with sterile PBS.</li>
	</ol>
	</li>
	<li>Add 1 mL DMEM into each well of a 12-well plate. Place a coverslide in each well.</li>
	<li>Seed 20,000 cells/well, drop by drop while shaking the plate at the same time for a homogenous distribution of cells on the coverslides. Keep cells in the CO2 incubator at 37 &#176;C and add the drug so that the total incubation time does not exceed 48 h, i.e., for 3 h Torin treatment, add the drug at 45 h post seeding.
	<ol>
		<li>For RACK1-LC3 interaction studies, Torin 1, rapamycin, and starvation were used as autophagy inducers. Cells were incubated with Torin 1 for 3 h (250 nM final concentration); with rapamycin for 16 h (200 nM final concentration); and with Earle&#39;s Balanced Salt Solution (EBSS) for 2 h in order to starve cells. 
		Note: Incubation in DMEM with 10% FBS was used as the control condition.</li>
	</ol>
	</li>
	<li>After 48 h of total incubation time, remove the media on the coverslides and wash them with 1 mL PBS.</li>
	<li>To fix the cells, incubate the cells with 1 mL ice cold sterile filtered 4% paraformaldehyde (PFA) in 1x PBS (pH 7.4), for 20 min at room temperature without any agitation. 
	Since PFA is sensitive to light, keep (important) the plate in the dark during the incubation period; covering the plate with aluminum foil may help. Moreover, PFA is toxic, therefore, work under a fume hood while fixing the cells. 
	Note: Keep in mind that the fixation step in immunofluorescence experiments should be optimized, as specific antibodies may work best using different fixation agents and conditions.</li>
	<li>After 20 min of PFA incubation, first remove the PFA solution on the coverslides and then wash each sample 3 times with 1 mL sterile filtered PBS. At this step, (important) wash the wells one by one so as not to let them dry. Avoid drying since it will interfere with subsequent reactions of the cells, change the cell morphology, and increase non-specific signals.</li>
	<li>When the wash is complete, keep the samples in 1 mL PBS on ice.</li>
	<li>Prepare the blocking solution: PBS with 0.1% Bovine Serum Albumin (BSA) and 0.1% saponin.</li>
	<li>Cover a 6-well plate with paraffin film. Ensure that the plate bottom is smooth after the paraffin film covering. Label the 6-well plate.</li>
	<li>Using tweezers, transfer the coverslides from the 12-well plates to the 6-well plates covered with paraffin film.</li>
	<li>Discard the PBS on coverslides and add 100 &#181;L of blocking solution onto each coverslide for permeabilization. Incubate the samples on ice for 30 min.</li>
	<li>Prepare the primary antibody in blocking solution. Prior to the experiments, optimize the antibody dilutions to define the best working concentrations for staining. 
	NOTE: For example, LC3 antibody is used at a 1:100 dilution in blocking solution.</li>
	<li>After the 30 min incubation, discard the blocking solution and wash the samples gently with 1 mL of PBS once.</li>
	<li>Add 100 &#181;L of primary antibody solution to each coverslide. Incubate the samples at room temperature for 1 h, on a shaker. 
	NOTE: Incubation time with the primary antibody should be optimized. Depending on the antibody, an overnight incubation at 4 &#176;C may be preferable. It is best to incubate on a shaker gently (100 rotations/min) to equally distribute the antibody on the coverslides.</li>
	<li>After incubation with the primary antibody, wash samples individually, 3 times with 1 mL PBS.</li>
	<li>Prepare 1:500 secondary antibody in blocking solution (optimizations of dilutions of the secondary antibody may be necessary). Here, anti-rabbit IgG Alexa Fluor 568 was used as a secondary anti-rabbit antibody.
	<ol>
		<li>Incubate the samples with the 100 &#181;L of secondary antibody solution for 1 h at room temperature on a shaker. Since the fluorophore-labeled secondary antibodies are sensitive to light, (important) keep the solution in the dark. Place the samples in the dark during and after the secondary antibody incubation.</li>
	</ol>
	</li>
	<li>After the secondary antibody incubation, wash the plate 3 times with PBS. 
	NOTE: Baed upon experience, some antibodies have high background signals that cannot be eliminated by the PBS washes. If dealing with such an antibody, after step 1.21, keeping the samples in PBS at 4 &#176;C for 6 h may help.
	<ol>
		<li>If needed, stain the cells with a DNA-binding dye, such as Hoechst, to mark the nuclei.</li>
	</ol>
	</li>
	<li>To stain more than one protein, apply the same procedure starting from step 1.16 for the second protein.
	<ol>
		<li>Make sure that there is no species reactivity between the antibodies when performing staining with more than one antibody (e.g., use mouse and rabbit antibodies). 
		Note: Here, RACK1 is stained as the second protein by using anti-RACK1 primary antibodies and anti-mouse IgG Alexa Fluor 488 secondary antibodies using the same concentrations mentioned above.</li>
	</ol>
	</li>
	<li>Prepare the mounting solution: 50% Glycerol in 1x PBS. Filter through a 22 &#181;m filter.</li>
	<li>Wipe the slides with ethanol using a lint-free wipe and add 10 &#181;L mounting solution to each slide. Using a tweezer, place the coverslides on each dropso that the side with the cells is in direct contact with the mounting medium. Discard excess mounting solution gently using lint-free wipes.</li>
	<li>Seal the coverslides with a transparent nail polish. First, stabilize the coverslides by adding drops of nail polish on the 4 edges and then seal them completely.</li>
	<li>Analyze the samples under a fluorescent or confocal microscope as soon as possible, since the fluorescent signal may bleach after a few hours.</li>
</ol>
2. Immunoprecipitation
<ol>
	<li>Detach and count the cells as described in steps 1.2 and 1.3.</li>
	<li>If working with HEK293T cells, seed 4 million cells for each condition in 15-cm Petri dishes. If working with N2A cells, seed 5 million cells in 15 cm Petri dishes. Incubate the cells in a CO2 incubator at 37 &#176;C until the end of the experiment.</li>
	<li>Treat the cells with the relevant drugs or conditions for a defined duration so that the total incubation time of the cells does not exceed 48 h. 
	Note: For the immunoprecipitation tests in this study, cells were treated for 3 h with Torin 1 (250 nM), 16 h with rapamycin (200 nM), and 2 h with EBSS. DMEM with 10% FBS was added to the cells as the control.</li>
	<li>After 48 h of total incubation time, remove the media and harvest the cells. Since HEK293T cells detach easily from the plate, washing the plate with ice cold PBS may be adequate for harvesting. However, N2A cells are more resistant to detachment; use cell scrapers to help harvest the cells.
	<ol>
		<li>Collect all cells in microcentrifuge tubes.</li>
	</ol>
	</li>
	<li>Centrifuge samples at 16,000 x g for 15 min at 4 &#176;C. Discard the supernatant and wash the pellet with 1 mL PBS by re-suspending it by pipetting.</li>
	<li>Repeat centrifuge step, but at the highest centrifuge speed at 4 &#176;C for 15 min. Discard the supernatant.</li>
	<li>Prepare Radio Immuno-Precipitation Assay (RIPA) buffer before starting the experiment. 
	NOTE: There are 3 types of RIPA buffer; choose according to the interaction intensity of the proteins being investigated (see formulas below).
	<ol>
		<li>If there is a strong protein-protein interaction, choose the regular RIPA buffer.</li>
		<li>If the interaction is weak, choose either mild RIPA or milder RIPA. 
		Note: The recipes of these buffers are in the following steps. All buffers that are used in immunoprecipitation tests should be supplemented with protease inhibitors.
		<ol>
			<li>Prepare regular RIPA Buffer using 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate in 50 mM Tris pH 8.</li>
			<li>Prepare mild RIPA Buffer using 1% NP-40, 150 mM NaCl, and 0.25% sodium deoxycholate in 50 mM Tris pH 7.5.</li>
			<li>Prepare the milder RIPA Buffer using 1% Triton-X 100, 137 mM NaCl, 1% glycerol, and 1 mM sodium orthovanadate in 50 mM Tris pH 7.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After choosing the appropriate RIPA buffer, supplement the buffer with protease inhibitors (RIPA+). Then, lyse the cells with 3x the volume of the cell pellet in RIPA+.</li>
	<li>Vortex the suspension for 15 s. Keep it on ice for 5 min. Repeat vortex and icing 5 times and then centrifuge the samples at 16,000 x g for 15 min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a clean microcentrifuge tube and centrifuge to remove remnant cell debris. Transfer supernatant to a clean tube.</li>
	<li>Dilute the protein lysates 1:50 and prepare a 96-well plate with blank and standards. Dilute the samples.
	<ol>
		<li>Mix samples with Bradford solution 1:20. Incubate for 15 min in the dark.</li>
		<li>Measure the optical density at 595 nm wavelength. Calculate the protein concentration using absorbance values for each sample.</li>
	</ol>
	</li>
	<li>The day before harvesting the cells, couple protein A Plus beads with antibodies that are specific to the protein of interest.
	<ol>
		<li>Cut the edge of a 200 &#181;L tip using scissors and use 25 &#181;L from the bead slurry for each condition.</li>
		<li>Wash the beads once with 1 mL PBS, once with 1 mL RIPA buffer, and once with 1 mL RIPA+. Carry out the wash steps at 3,300 x g, 4 &#176;C for 1 min.</li>
		<li>After the last wash, add 300 &#181;L of RIPA+ on the beads and add 1.5 &#181;g antibody that is specific to the protein to be pulled down.</li>
		<li>Incubate the tubes overnight at 4 &#176;C on a rotator (20 rotations/min). 
		Note: Here, ATG5 was immunoprecipitated using specific anti-ATG5 antibodies.</li>
	</ol>
	</li>
	<li>After the overnight incubation of beads with the antibody, wash the beads once with PBS, once with RIPA, and once with RIPA+ as described in step 2.12.2.</li>
	<li>Add 2 mg of protein lysate on the beads for each condition and complete the total volume to 300 &#181;L with RIPA+ buffer/tube. Incubate the tubes overnight on a rotator at 4 &#176;C.</li>
	<li>After the incubation, wash the beads as previously described (step 2.12.2).
	<ol>
		<li>After the last wash, use a smaller pipet tip to remove all the supernatant without touching the beads.</li>
		<li>Add 10 &#181;L of 3x loading dye: 6% SDS, 30% Glycerol, 16% &#946;-Mercaptoethanol, and 0.1% Bromophenol blue in 1M Tris-HCl pH 6.8.</li>
	</ol>
	</li>
	<li>Prepare the input controls using at least 100 &#181;g protein lysate for each condition. Equalize the sample volumes using water and add the appropriate volume of 3x loading dye.</li>
	<li>Boil samples at 95 &#176;C for 10 min. 
	Note: Usually when boiling the samples, the caps of the tubes can open spontaneously and samples are lost. Avoid this by holding the caps closed with a specific cap blocker.</li>
	<li>Spin down the samples and load onto SDS-PAGE gels to separate the proteins according to their sizes using standard protocols. 
	Note: In this study on RACK1 and ATG5, samples were run through 12% polyacrylamide gels at 80 V for 30 min, and then at 120 V for approximately 90 min. However, for smaller proteins such as LC3, using 15% polyacrylamide gels is more suitable.</li>
	<li>After the gel running is complete, transfer the proteins to nitrocellulose or PVDF membranes using either wet or semi-dry transfer methods. Then, block the membranes by incubation in 5% non-fat milk in PBS with 0.05% Tween 20 (PBST) at room temperature for 1 h. Wash membranes 3 times with PBST for 5 min.</li>
	<li>Incubate the membranes with primary antibodies at working concentrations for 1 h at room temperature. For primary antibody dilutions, use BSA containing red solutions (5% BSA Cohn V Fraction, 0.02% sodium azide in PBST, pH 7.5; add Phenol red added as a pH marker) to permit the reuse of antibodies. 
	NOTE: For example, dilute ATG5 antibody 2,000x in the red solution.
	<ol>
		<li>At the end of the incubation time, wash the membranes 3 times with PBST.</li>
	</ol>
	</li>
	<li>Incubate the membranes with HRP-conjugated secondary antibodies that were prepared in 5% non-fat milk solution (here, anti-rabbit IgG at 1:10.000 was used). Then wash the membranes 3x with PBST for 5 min.</li>
	<li>Make the ECL solution: 25 mM luminol, 9 mM coumaric acid, 3 x 10-4 % H2O2 in 70 mM Tris-HCl pH 8.8. Use fresh H2O2 for each experiment. 
	NOTE: The reaction starts after H2O2 addition and it continues for approximately 20 min.
	<ol>
		<li>Incubate the membranes and X-ray films for 20 min in ECL. Develop and fix them in a dark room.</li>
	</ol>
	</li>
	<li>To check the co-immunoprecipitation of proteins with close or overlapping molecular weights, it may be necessary to strip the antibodies off the membranes by incubating the membranes at 60 &#176;C for 30 min in the stripping buffer: (25 mM Tris-HCl pH 2 and 1% SDS).</li>
	<li>Repeat steps 2.20 to 2.23 to check for co-immunoprecipitation using different antibodies. For example, following ATG5 detection and stripping of the membranes, RACK1 protein was tested on the same membranes using an anti-RACK1 primary antibody (1:1,000) and secondary antibodies (1:10,000). &#946;-Actin was used as the loading control; and IgG signal was used as the loading control for immunoprecipitated samples.</li>
</ol>

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The authors declare that they have no competing financial interests.

Immunoprecipitation and immunofluorescence techniques are crucial for the study of protein-protein interactions. Although these two techniques are commonly used and well-established, several criteria should be considered to define the quality of experiments while using these techniques.
First, primary antibodies that are used in these tests should be specific to the proteins of interest. To ensure this, use shRNA knockdowns or knockout cells to test the specificity of the antibody in question....

Macroautophagy (autophagy, herein) is a cellular stress mechanism that is characterized by the sequestration of bulk cytoplasm, proteins, and organelles in double membrane vesicles called autophagic vesicles. Through fusion of the outer layer of the double membrane, autophagic vesicles and their cargo are delivered to lysosomes and degraded therein1. Autophagy occurs at low basal levels in all cell types and in all organisms, performing homeostatic functions such as protein degradation and organelle (e.g., mitochondria) turnover. Under conditions leading to cellular stress, such as starvation, autophagy is rapidly upregulated and allows the cell to maintain energy levels and basic metabolism1,2,3.
Around 30 autophagy genes have been cloned from the yeast and their protein products were shown to play a role in various stages of the autophagic process, including vesicle nucleation, expansion, vesicle fusion to late endosome/lysosome, and cargo degradation4,5. Orthologs of the majority of these genes have been identified and studies in various organisms confirmed preservation of their cellular functions6. Studies in the last decade showed that several autophagy-related protein complexes and protein-protein interactions exist and that they govern autophagy pathways in an intricate and controlled manner. Intersections, backups, feedback, and feedforward mechanisms exist, and they allow the cell to coordinate autophagy with other related events (such as vesicular secretion, lysosome biogenesis, endosomal sorting and transport7, etc.) In an unbiased yeast-two hybrid screen using the autophagy protein ATG5 as a bait, (ATG5 is a key autophagy protein involved in the E2-like conjugating system that mediates LC3 lipidation in starvation induced autophagy), we have identified Receptor Activated C-Kinase 1 (RACK1; GNB2L1) as a strong interactor and a novel autophagy component8. Importantly, the screen showed that the ATG5-RACK1 interaction was indispensable for autophagy induction by classical autophagy inducers (i.e., starvation and mTOR inhibition).
Immunofluorescence-based methods are commonly used to monitor protein-protein interactions. These techniques are mainly antibody-based, and help to visualize interactions and confirm cellular localizations. In this technique, fluorescent tag conjugated antibodies that are specific to proteins of interest are generally used for specific staining. Each protein may be labeled with antibodies coupled to different fluorescent dyes. Using protein-specific antibodies, an overlap in the signal when images are merged indicates the co-localization of proteins under confocal microscopy. The technique is applicable to cells or even tissues. Immunofluorescence techniques provide clues about interaction dynamics, and help identify the size and distribution of protein complexes, while tracking general changes in cellular morphology under different conditions9. Immunoprecipitation is another commonly used antibody-based technique that allows for the analysis of interactions between given proteins10. Using this technique, proteins of interest are isolated from cells or tissue extracts using specific antibodies, resulting in the precipitation of proteins that are in a complex or in contact with a protein of interest. Co-immunoprecipitation, where the protein and its co-interactor are detected, reveals not only the interaction between the two proteins, but can measure its strength of interaction under different circumstances11.
This protocol describes in detail key techniques that were used to confirm and characterize ATG5-RACK1 and RACK1-LC3 interaction. The focus is on immunofluorescence and immunoprecipitation techniques, with emphasis of critical steps and pitfalls for autophagy research, as well as troubleshooting suggestions.

<table><tbody><tr><td>Trypsin EDTA Solution A</td><td>Biological Industries&amp;nbsp;</td><td>BI03-050-1A</td><td></td></tr><tr><td>PBS</td><td>GE Healthcare</td><td>SH-30256.01</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Sigma&amp;nbsp;</td><td>5671</td><td></td></tr><tr><td>DMEM (low glucose)</td><td>Sigma</td><td>5546</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma</td><td>Z359629-1EA</td><td></td></tr><tr><td>coverslides</td><td>Jena Bioscience</td><td>CSL-103</td><td></td></tr><tr><td>slides</td><td>Isolab</td><td>I.075.02.005</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma&amp;nbsp;</td><td>P8920</td><td></td></tr><tr><td>Torin</td><td>Tocris</td><td>4247</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>VWRSAD2650</td><td></td></tr><tr><td>EBSS</td><td>Biological Industries&amp;nbsp;</td><td>BI02-010-1A</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>15812-7</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>&amp;nbsp;A4503</td><td></td></tr><tr><td>Saponin</td><td>Sigma</td><td>84510</td><td></td></tr><tr><td>LC3 Antibody</td><td>Sigma</td><td>L7543</td><td></td></tr><tr><td>Anti-Rabbit IgG Alexa Fluor 568</td><td>&amp;nbsp;Invitrogen</td><td>A11011</td><td></td></tr><tr><td>RACK1 Antibody</td><td>Santa Cruz Biotechnology</td><td>&amp;nbsp;sc-17754</td><td></td></tr><tr><td>Anti-Mouse IgG Alexa Fluor 488</td><td>&amp;nbsp;Invitrogen</td><td>A11001</td><td></td></tr><tr><td>NP-40</td><td>Applichem</td><td>A16694.0250</td><td></td></tr><tr><td>Sodium Chloride</td><td>Applichem</td><td>A9242.5000</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>30970</td><td></td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Biochemika</td><td>A2572</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Triton-X&amp;nbsp;</td><td>Applichem</td><td>4975</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma</td><td>450243</td><td></td></tr><tr><td>ATG5 Antibody</td><td>Sigma</td><td>A0856</td><td></td></tr><tr><td>Glycerol</td><td>Applichem</td><td>A4453</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>Applichem</td><td>A1108.0250</td><td></td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Applichem</td><td>A3640.0005</td><td></td></tr><tr><td>Non-Fat milk</td><td>Applichem</td><td>A0830</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Sodium Azide</td><td>Riedel de Haen</td><td>13412</td><td></td></tr><tr><td>Phenol red&amp;nbsp;</td><td>Sigma</td><td>114537-5G</td><td></td></tr><tr><td>anti-rabbit IgG , HRP conjugated</td><td>Jackson Immuno.&amp;nbsp;</td><td>1110305144</td><td></td></tr><tr><td>Luminol</td><td>Fluka</td><td>9253</td><td></td></tr><tr><td>Coumeric Acid</td><td>Sigma</td><td>C9008</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Merck</td><td>K35522500604</td><td></td></tr><tr><td>anti mouse IgG, HRP conjugated</td><td>Jackson Immuno.</td><td>115035003</td><td></td></tr><tr><td>&amp;beta;-Actin Antibody</td><td>Sigma&amp;nbsp;</td><td>A5441</td><td></td></tr><tr><td>Normal rabbit serum</td><td>Santa Cruz Biotechnology</td><td>sc-2027</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma</td><td>&amp;nbsp;R0395</td><td></td></tr><tr><td>Protein A-Agarose Beads</td><td>Santa Cruz Biotechnology</td><td>sc-2001</td><td></td></tr><tr><td>fetal bovine serum&amp;nbsp;</td><td>Biowest</td><td>S1810-500</td><td></td></tr><tr><td>penicillin/streptomycin solution</td><td>Biological Industries&amp;nbsp;</td><td>03-031-1B</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Biological Industries&amp;nbsp;</td><td>BI03-020-1B</td><td></td></tr><tr><td>Bradford Solution</td><td>Sigma</td><td>6916</td><td></td></tr><tr><td>Nitocellulose membrane</td><td>GE Healthcare</td><td>A10083108</td><td></td></tr><tr><td>X-ray Films</td><td>Fujifilm</td><td>47410 19289</td><td></td></tr><tr><td>Protease inhibitor</td><td>Sigma</td><td>P8340</td><td></td></tr></tbody></table>

study of protein-protein interactions in autophagy research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

In Figure 1 an example of a co-localization result obtained using this protocol is shown. A figure from our recent paper is presented8. Here, endogenous RACK1 protein was stained in green, while endogenous LC3 was stained in red. The yellow dots that are observed in the merged pictures indicate sites of overlap between the green and red signals. Thus the yellow dots represent the partial co-localization of these two proteins.
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Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

Study of Protein-protein Interactions in Autophagy Research

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

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Biology

9.1K Views.  Sabanci University. Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics. 

Video: Study of Protein-protein Interactions in Autophagy Research

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Cancer Research

Assessing LC3-II-Positive EV Release in Autophagy with Impaired Lysosome Fusion

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.
To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf ...

Biochemistry

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Investigating ATG9A Localization in Cells

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

Author Spotlight: Imaging ATG9A, a Multi-Spanning Membrane Protein  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

High-Throughput Screening Analysis for 4B Cysteine Peptidase Autophagy Inhibitor

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells 

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...

Study of Protein-protein Interactions in Autophagy Research

Summary

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Chapters in this video

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Activating Autophagy by Aerobic Exercise in Mice

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Imaging ATG9A, a Multi-Spanning Membrane Protein

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase