Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Summary

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

Abstract

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.

Introduction

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 “clock genes” 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 b....

Protocol

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

1. To detect daily rhythms in protein turnover, house mice (male or female C57BL/6J, 4−8 week old, 20−25 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’s internal biological clock), house mice under consta....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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