This work was funded by RO1HL135846 and a Children&#8217;s Development Institute grant (PD-II-2016-529).

The protocol described here was approved by the Washington University in St. Louis Animal Care and Use Committee (IACUC).
1. Mouse Housing and Experimental Design
<ol>
	<li>To detect daily rhythms in protein turnover, house mice (male or female C57BL/6J, 4&#8722;8 week old, 20&#8722;25 g) under standard 12 h light/dark cycles with food provided ad libitum. To avoid stressing the animals, acclimate mice for at least one week in the facility prior to use and avoid single housing. For proving that rhythms in protein catabolism are truly circadian (i.e., driven by the animal&#8217;s internal biological clock), house mice under constant dark conditions, taking care to avoid exposing the mice to exogenous light. 
	NOTE: For details about mouse housing for circadian rhythm experiments see the following reference11.</li>
	<li>For each time point, allocate three mice as sham controls and at least three mice for each type of protease inhibitor used (leupeptin for autophagic flux and bortezomib for proteasomal flux measurements). Plan for six time points evenly spaced at 4 h intervals across the day/night cycle to obtain tissue samples. 
	NOTE: A 24 h, 6 time point experiment with phosphate-buffered saline (PBS), leupeptin, and bortezomib will require a total of 54 mice (3 per treatment x 3 treatments x 6 time points).</li>
</ol>
2. Preparation of Homogenization Solution, Inhibitor Stock Solutions, and Vials for Mouse Liver Collection
<ol>
	<li>Prepare fresh homogenization buffer (HB) and keep on ice: 10 mM Tris-HCl pH 7.5, 250 mM sucrose, 5 mM EDTA pH 8.0, and 1 protease inhibitor tablet per 100 mL. 
	NOTE: Approximately 10 mL of HB is needed per mouse.</li>
	<li>For downstream processing of biological samples, each sample requires a 15 mL conical tube to hold the crude homogenate, a 15 mL tube to hold the post-nuclear supernatant, two 1.5 mL microcentrifuge tubes to hold the 3,000 x g and 20,000 x g pelleted material, respectively, and a 1.5 mL microcentrifuge tube to hold the cytoplasmic fraction. Add 7 mL of cold HB to each tube intended for crude homogenate and keep on ice.</li>
	<li>Prepare a 2 mg/mL stock solution of leupeptin hemi-sulfate in sterile PBS. Also prepare a 50 mg/mL solution of bortezomib in dimethyl sulfoxide (DMSO). Aliquot and store the solutions at -80 &#176;C.</li>
</ol>
3. Protease Inhibitor Administration
<ol>
	<li>Thaw leupeptin (2 mg/mL) and bortezomib (50 mg/mL) stock solutions to room temperature 15 min prior to each time point. The leupeptin stock is ready for injection. Dilute the bortezomib in sterile PBS to yield a 50 &#181;g/mL working solution. 
	NOTE: Bortezomib is light sensitive, so protect the working stock from light until use.</li>
	<li>Weigh mice and perform intraperitoneal injections of &#8220;sham&#8221; PBS (0.5 mL), leupeptin (40 mg/kg), or bortezomib (1.6 &#181;g/kg). Return mice to appropriately marked cages grouped by the type of injection they received. Record the time the mice are treated or manipulated in a standardized table (see Supplemental File &#8220;Sample Data&#8221;). 
	NOTE: A dose calculator is included as an aid in the Supplemental File &#8220;Sample Data&#8221;. It is important to perform injections expeditiously and in the same order from timepoint to timepoint to minimize variability.</li>
</ol>
4. Tissue Acquisition and Storage
<ol>
	<li>Two hours after injection, euthanize mice one at a time in accordance with the institutionally approved IACUC protocol. For example, anesthetize mice then euthanize by cervical dislocation. Proceed to the dissection step immediately following euthanasia. 
	NOTE: Ensure complete anesthesia before cervical dislocation by pinching front paws without an observable reflex.</li>
	<li>Remove the left lobe of the liver by making a 1.5 to 2.0 cm incision with Metzenbaum scissors on the mouse&#8217;s ventral abdomen. Externalize the left lobe of the liver and excise it with surgical scissors. Submerge the liver sample in 7 mL of ice-cold HB in an appropriately labeled 15 mL conical tube. Keep the sample on ice throughout processing. 
	NOTE: The samples can be kept on ice or at 4 &#176;C overnight before proceeding. If standard markers of macroautophagy and proteasomal activity are the only intended readouts, liver samples can be flash frozen in liquid nitrogen and stored at -80 &#176;C for later processing. To examine other targets of degradation (e.g., via proteomics), process the samples without freezing.</li>
	<li>Proceed to euthanize and dissect the next mouse until all samples are acquired. Process mice in the same group order they were injected and record the duration of this step (see Supplemental File &#8220;Sample Data&#8221;). Each mouse should be processed in under 5 min to limit difference in the exposure times for the injected inhibitors.</li>
</ol>
5. Biochemical Fractionation of Liver Samples
NOTE: Figure 1B shows the fractionation scheme.
<ol>
	<li>Transfer each liver sample and 7 mL of HB into a 15 mL capacity Dounce homogenizer. Homogenize the sample with 10 strokes of the loose piston and 15 strokes of the tight piston. Return the resultant crude homogenate to the 15 mL conical tube it originated from. Repeat until all liver samples have been homogenized.</li>
	<li>Spin crude homogenate samples at 700 x g for 10 min at 4 &#176;C to pellet nuclei and debris. Transfer the top 4 mL of post-nuclear supernatant (S1) to a fresh 15 mL conical tube.</li>
	<li>Determine the protein concentration of fraction S1 using Bradford or bicinchoninic acid&#160;(BCA) assays according to the manufacturer&#8217;s instructions. Next equalize the concentrations of all samples to 2.1 mg/mL or to the desired concentration by adding fresh HB to S1 where needed. The normalized samples are the &#8220;Total Protein&#8221; (Tot) fraction. For downstream analytical purposes and quality improvement, aliquot 500 &#181;L of the Tot fraction and store at -80 &#176;C.</li>
	<li>Transfer 1.5 mL of the remaining Tot fraction into a microcentrifuge tube and spin at 3,000 x g for 15 min at 4 &#176;C. Transfer 1 mL of the resultant supernatant (S2) to a fresh microcentrifuge tube and set on ice.</li>
	<li>Aspirate the remaining supernatant and wash the 3,000 x g pellet (3KP) twice with 1.5 mL of cold HB. The 3KP pellet can be stored dry at -80 &#176;C if downstream proteomics are anticipated. If only western blotting is anticipated, resuspend the 3KP pellet in 200 &#181;L of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Table of Materials) and boil at 95 &#176;C for 5 min.</li>
	<li>Spin the S2 fraction at 20,000 x g for 20 min at 4 &#176;C. Transfer the resultant supernatant (S3) to a fresh microcentrifuge tube. S3 is the cytoplasmic (Cyto) fraction. For SDS-PAGE, combine 150 &#181;L of Cyto fraction with 50 &#181;L of 4x SDS-PAGE sample buffer and boil at 95 &#176;C for 5 min.</li>
	<li>Aspirate the remaining supernatant from the 20,000 x g pellet (20KP) and wash twice with 1.5 mL of cold HB. The 20KP pellet can be stored dry at -80 &#176;C if downstream proteomics are anticipated. If only western blotting is anticipated, resuspend the 20KP pellet in 100 &#181;L of SDS-PAGE sample buffer and boil at 95 &#176;C for 5 min.</li>
</ol>
6. Western Blotting Readout
<ol>
	<li>In order to quantify macroautophagic and proteasomal flux via western blotting, compose a stock solution of purified GST-LC3b (2 &#181;g/mL), p62-His6 (2 &#181;g/mL), and Lys48 linked polyubiquitin (K48-Ub, 50 &#181;g/mL) in 1x SDS-PAGE sample buffer. After boiling at 95 &#176;C for 5 min, aliquot and store at -80 &#176;C for future use.</li>
	<li>At the time of SDS-PAGE, generate a 6-point standard curve of the standard protein mixture by serially diluting 1:3 in 1x SDS-PAGE sample buffer. Load 10 &#181;L of each standard curve sample onto a 26 well 4&#8722;12% SDS-PAGE midi-gel, followed by prestained protein standards (Table of Materials), a commercial molecular weight standard.</li>
	<li>For measuring autophagic flux, load 12 &#181;L of 3KP sample into each well. 
	NOTE: Assuming sham, bortezomib, and leupeptin treatments were done and assuming 3 mice per treatment, each time point should consist of 9 samples. Therefore, each midi-gel can accommodate 2 time points plus the standard curve and a typical time series experiment will require 3 midi-gels for readout.</li>
	<li>For measuring proteasomal flux, load 12 &#181;L of Cyto fraction into each well.</li>
	<li>Separate protein samples by SDS-PAGE and transfer to polyvinylidene fluoride (PVDF) membranes using standard protocols. If western blotting for LC3b is anticipated, transfer gels using 20% methanol-containing transfer buffer. Otherwise, use 10% methanol-containing transfer buffer as this will enable more efficient transfer of high molecular weight protein species.</li>
	<li>Perform western blotting using standard protocols. For analyzing macroautophagic flux, blot membranes containing 3KP samples with anti-p62 or anti-LC3b antibodies (1:1,000) overnight at 4 &#176;C. For analyzing proteasomal flux, blot membranes containing Cyto samples with anti-Lys48 specific polyubiquitin (1:1,000) overnight at 4 &#176;C.</li>
	<li>Image western blots using standard secondary antibodies and imaging devices. Image all membranes that constitute a given time series experiment together.</li>
</ol>
7. Data Analysis
NOTE: See Supplemental File &#8220;Sample Data&#8221;.
<ol>
	<li>To perform densitometry on western blot samples, use standard graphics software, Image Studio, or alternatively ImageJ.</li>
	<li>Perform densitometric measurements on bands of interest for both the standard curve and the experimental samples. In Image Studio, using p62 as an example, draw a long rectangle encompassing protein monomer at the bottom and extending to the top of the membrane. Copy, paste, and move the rectangle to the remaining samples. It is important to keep the rectangle used for quantification consistent between samples.</li>
	<li>Save the quantification to a spreadsheet by pressing Report and Launch Spreadsheet at the bottom right of the analysis window.</li>
	<li>Generate a densitometric standard curve with spreadsheet using the serially diluted standard sample and use either linear or polynomial regression to obtain a best fit standard curve equation. Using this equation, extrapolate the quantity of p62, LC3b-II or Lys48-poly Ub in the experimental samples.</li>
	<li>To obtain flux measurements, subtract the extrapolated protein quantity of each inhibitor-treated sample from the average value of the PBS samples from the same time point (see Supplemental File &#8220;Sample Data&#8221;).</li>
	<li>Evaluate the statistical significance of temporal variation in protein turnover using 1-way ANOVA. This can be done using standard spreadsheet software.</li>
</ol>

<ol>
	<li>Green, C. B., Takahashi, J. S., Bass, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+meter+of+metabolism.">The meter of metabolism.</a> Cell. 134 (5), 728-742 (2008).</li><li>Ma, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS+suppresses+expression+of+asialoglycoprotein-binding+protein+through+TLR4+in+thioglycolate-elicited+peritoneal+macrophages.">LPS suppresses expression of asialoglycoprotein-binding protein through TLR4 in thioglycolate-elicited peritoneal macrophages.</a> Glycoconjugate Journal. 24 (4-5), 243-249 (2007).</li><li>Ryzhikov, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+Rhythms+Spatially+and+Temporally+Organize+Autophagy.">Diurnal Rhythms Spatially and Temporally Organize Autophagy.</a> Cell Reports. 26 (7), 1880-1892 (2019).</li><li>Martinez-Lopez, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-wide+Benefits+of+Intermeal+Fasting+by+Autophagy.">System-wide Benefits of Intermeal Fasting by Autophagy.</a> Cell Metabolism. 26 (6), 856-871 (2017).</li><li>Desvergne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+proteasome+activity+and+accumulation+of+oxidized+protein+in+human+embryonic+kidney+HEK+293+cells+and+primary+dermal+fibroblasts.">Circadian modulation of proteasome activity and accumulation of oxidized protein in human embryonic kidney HEK 293 cells and primary dermal fibroblasts.</a> Free Radical Biology and Medicine. 94, 195-207 (2016).</li><li>Levine, B., Mizushima, N., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+immunity+and+inflammation.">Autophagy in immunity and inflammation.</a> Nature. 469 (7330), 323-335 (2011).</li><li>Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+protein+degradation:+from+a+vague+idea+thru+the+lysosome+and+the+ubiquitin-proteasome+system+and+onto+human+diseases+and+drug+targeting.">Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting.</a> Cell Death &amp; Differentiation. 12 (9), 1178-1190 (2005).</li><li>Collins, G. A., Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Logic+of+the+26S+Proteasome.">The Logic of the 26S Proteasome.</a> Cell. 169 (5), 792-806 (2017).</li><li>Haspel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+macroautophagic+flux+in+vivo+using+a+leupeptin-based+assay.">Characterization of macroautophagic flux in vivo using a leupeptin-based assay.</a> Autophagy. 7 (6), 629-642 (2011).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).</a> Autophagy. 12 (1), 1-222 (2016).</li><li>Eckel-Mahan, K., Sassone-Corsi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotyping+Circadian+Rhythms+in+Mice.">Phenotyping Circadian Rhythms in Mice.</a> Current Protocols in Mouse Biology. 5 (3), 271-281 (2015).</li><li>Hughes, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+Genome-Scale+Analysis+of+Biological+Rhythms.">Guidelines for Genome-Scale Analysis of Biological Rhythms.</a> Journal of Biological Rhythms. 32 (5), 380-393 (2017).</li></ol>

The authors have nothing to disclose.

Our protocol describes a technically straightforward means of measuring biological rhythms in protein turnover in mice using commonly available molecular biology equipment. Because of the length of time series experiments and the number of biological samples involved, it is important to be consistent across the entire experiment regarding how the mice are injected, the timing of tissue acquisition and the biochemical processing of samples. The injection, euthanasia, and cervical dislocation steps may require operator pra...

Circadian rhythms refer to daily, predictable variations in biological function that are apparent throughout nature. They exist at every biological scale, from macroscopic behaviors like sleep-wake cycles, to molecular phenomena like the rhythmic abundance of biomolecules. In recent years, research into circadian rhythms has been transformed by the discovery of &#8220;clock genes&#8221; that are critical for circadian rhythm generation. Studies in clock gene knockout mice have revealed a central role for circadian rhythms in temporally organizing core cellular processes such as metabolism1. Among the ways circadian rhythms make this happen is by imparting a temporal structure to protein catabolism.
Several groups including ours have shown that the two major avenues for cellular protein catabolism, autophagy and the ubiquitin-proteasome system, are subject to diurnal rhythms2,3,4,5. Autophagy represents the lysosome-dependent arm of protein catabolism in which proteins of interest are delivered to this degradative organelle either through the construction of a novel vesicle (macroautophagy) or through direct translocation though a channel (chaperone mediated autophagy)6. The ubiquitin-proteasome system is the main non-lysosomal pathway, where proteins are poly-ubiquitinated and then fed into the proteasome, a macromolecular degradative machine found throughout the cytoplasm and nucleus7,8. Rhythms in autophagic and proteasomal activity are important because they likely play a role in cellular housekeeping. As a result, it is valuable to have a standardized procedure that can detect daily oscillations in protein catabolism that is compatible with pre-clinical disease models.
Here, we provide our protocol for quantifying diurnal variations in autophagic flux in mouse liver, which has served as the basis for work in our laboratory3,9. Our method is classified as a &#8220;turnover assay&#8221;10, an approach used by numerous groups to measure proteolytic activity (or flux). In this approach, protease inhibitors specific to lysosomes or proteasomes are administered to mice and then tissue samples are obtained after a fixed time interval. In parallel, tissue samples are obtained from mice subjected to sham injections. The tissue samples are homogenized and then biochemically separated to obtain the lysosome-enriched and cytoplasmic fractions. These fractions are then analyzed in parallel via western blotting using antibodies specific to macroautophagy markers (LC3b and p62) or proteasomal substrates (poly-ubiquitinated protein). Over time, animals injected with protease inhibitors accumulate proteins that would normally have been recycled. As a result, the rate of turnover is inferred by comparing the abundance of marker proteins in the protease-inhibitor treated samples to the sham-treated samples. By repeating this method at fixed time intervals across the day it is possible to reconstruct circadian variations in proteolysis (Figure 1A).

<table>
	<tbody>
		<tr>
			<td>4x SDS PAGE Sample Buffer</td>
			<td>Invitrogen</td>
			<td>Cat# NP0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bortezomib</td>
			<td>EMD Millipore</td>
			<td>Cat# 5.04314.0001; CAS: 179324-69-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Studio</td>
			<td>LICOR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane 0.45 micron</td>
			<td>Merck Millipore Ltd</td>
			<td>Cat# IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>Cat#8081S; RRID:AB_10859893</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3a</td>
			<td>Boston Biochem</td>
			<td>Cat# UL-430</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3b antibody</td>
			<td>Novus</td>
			<td>Cat#NB100-2220; RRID:AB_10003146</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3b antibody</td>
			<td>Cell Signaling Technology</td>
			<td>Cat#2775; RRID:AB_915950</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>Cat# L2884; CAS: 103476-89-7</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# WG1403BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>P62-his</td>
			<td>Novus</td>
			<td>Cat# NBP1-44490</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein All Blue Prestained Protein Standards</td>
			<td>Bio-Rad</td>
			<td>Cat# 1610373</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-p62/SQSTM1</td>
			<td>Millipore-Sigma</td>
			<td>Cat#P0067; RRID:AB_1841064</td>
			<td></td>
		</tr>
		<tr>
			<td>rhPoly-Ub WT (2-7) (K48)</td>
			<td>Boston Biochem</td>
			<td>Cat# UC-230</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE Midi-size Gels</td>
			<td>Invitrogen</td>
			<td>Cat# WG1403</td>
			<td></td>
		</tr>
		<tr>
			<td>SIGMAFAST Protease Inhibitor Tablets</td>
			<td>Millipore-Sigma</td>
			<td>Cat# S8830</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring diurnal rhythms in autophagic and proteasomal flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

Representative data are presented in Figure 2A,B, and the quantification of these data are provided in Figure 2C,D (see also Supplemental File &#8220;Sample Data&#8221;). For simplicity, we have not depicted loading controls in Figure 2 but these should be obtained in parallel. Typically, western blots against &#946;-actin are used for this purpose, but a total protein stain (such a...

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Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

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4.5K Views.  Washington University School of Medicine. We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

Video: Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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.

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

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 ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...