Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Summary

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Abstract

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton – a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

Introduction

IFs are a family of proteins that in humans are encoded by 73 genes and categorized into six major types: types I-IV are cytoplasmic (e.g. epithelial and hair keratins (K), myocyte desmin, neurofilaments, glial fibrillary acidic protein (GFAP), and others); type V are the nuclear lamins; and type VI are IFs in the eye lens¹. In terms of their molecular organization, IF proteins have three common domains: a highly conserved coiled-coil "rod" domain, and globular "head" and "tail" domains. IF protein tetramers assemble to form short filament precursors, which are ultimately incorporated into mature filaments that ....

Protocol

The protocol is approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina.

1. Preparations

1. Prepare Triton-X buffer (1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA), bring up volume in phosphate-buffered saline (PBS), pH 7.4). To make 500 mL: stir 5 mL each of Triton X-100 and 500 mM EDTA into 490 mL of PBS, pH 7.4. Store Triton-X buffer solution at 4 °C.
2. Prepare High Salt Buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, 0.5% Triton X-100, bring up volume in double distilled (dd) H₂O). To make 500 mL: stir 10 mL of 0.5 ....

Results

A new rapid method for high salt-based extraction of IF proteins from multiple mouse tissues using lysing matrix.

The traditional method^25,26 of isolating the bulk of the intermediate filament protein fraction from epithelial tissue was modified here to include 9 different organs and a more rapid procedure for tiss.......

Discussion

Methods that enable biochemical characterization of IF proteins can be useful to understand numerous pathophysiological phenomena in mammalian systems, since IF proteins are both markers and modulators of cellular and tissue stress²⁹. The principle behind the current method is based on the initial procedures developed in the 1970s and 1980s to isolate, separate and reconstitute IF proteins from cells and tissues, generally employing low and high salt solutions and Triton-X100 detergent

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dynabeads Protein G	ThermoFisher Scientific	10009	immunoprecipitation beads
PBS	ThermoFisher Scientific	10010049	for buffers
Purelink RNA mini kit	ThermoFisher Scientific	12183018	RNA extraction from tissue
Purelink DNAse set	ThermoFisher Scientific	12185010A	on column DNA digestion
Dynamag-2	ThermoFisher Scientific	12321D	magnet for use with dynabeads
GelCode Blue Stain Reagent	ThermoFisher Scientific	24592	mass spectrometry-compatible gel stain
Pierce ECL Western Blotting Substrate	ThermoFisher Scientific	32106	for use in western blot
High Capacity cDNA reverse transcription kit	ThermoFisher Scientific	4368813	for use in gene expression analysis
Proflex 3 x 32-well PCR System	ThermoFisher Scientific	4484073	PCR system
PVDF transfer membrane	ThermoFisher Scientific	88520	for western blot
Power Up SYBR master mix	ThermoFisher Scientific	A25778	for qPCR analysis
RNAlater	ThermoFisher Scientific	AM7020	solution for tissue storage prior to RNA isolation
Novex 4-20% Tris Glycine Gel	ThermoFisher Scientific	XP04205BOX	Precast protein gel
Anti-Keratin 8 antibody (TS1)	ThermoFisher Scientific	MA514428	for western blot detection of K8
Anti-Vimentin	ThermoFisher Scientific	MA511883	for western blot detection of vimentin
Tris Glycine Transfer Buffer (25x)	ThermoFisher Scientific	LC3675	for wet transfer of protein gels
2x SDS Sample Buffer	ThermoFisher Scientific	LC2676	for preparing protein gel samples
Tris Glycine SDS Running Buffer (10x)	ThermoFisher Scientific	LC26755	for running protein gels
Lysing beads - Matrix D	MP Biomedicals	116913100	Lysis beads and matrix tubes for tissue disruption and RNA extraction
Lysing beads - Matrix SS	MP Biomedicals	116921100	Lysis beads and matrix tubes for tissue disruption and protein extraction
NanoDrop Lite Spectrophotometer	ThermoFisher Scientific	ND-LITE	measurement of protein and nucleic acid
Precellys 24 homogenizer	Bertin Instruments	EQ03119	Automated tissue homogenizer