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 shape dynamic cytoskeletal and nucleoskeletal structures involved in mechanical protection², stress sensing^3,4, regulation of transcription⁵ and growth, and other critical cellular functions^1,6,7.

The functional importance of the IF system is highlighted by the existence of many human diseases caused by missense mutations in IF genes, including neuropathies, myopathies, skin fragility disorders, metabolic dysfunctions, and premature aging syndromes⁸. Some IF gene mutations do not cause, but predispose their carriers to disease progression, such as the simple epithelial keratins in liver disease⁹. The latter is due to the critical stress-protective functions of IFs in epithelia. IFs in general are among the most abundant cellular proteins under basal conditions, but are further strongly induced during various types of stress¹⁰. For example, recent studies evaluating proteome-wide changes in the nematode C. elegans demonstrated that multiple IFs are highly upregulated and prone to aggregation during organismal aging^11,12. Since maintenance of a proper IF structure is essential for cellular resistance to various forms of stress¹⁰, IF aggregation may also contribute to the functional decline during aging. However, organismal-level studies examining multiple mammalian IF proteins across different tissues undergoing stress are lacking.

IFs are highly dynamic structures that adapt to meet cellular demands. Keratins, for example, undergo a biosynthesis-independent cycling between soluble (non-filamentous) and insoluble (filamentous) protein pool¹³. Under normal physiologic conditions approximately 5% the total K8/K18 pool can be extracted in detergent-free buffer, in comparison to approximately 20% that can be solubilized in the non-ionic detergent Nonidet P-40, which is biochemically comparable to Triton-X100^14,15. During mitosis there is a notable increase in the solubility of simple-type epithelial K8 and K18¹⁴, which is less apparent in epidermal keratins but more apparent in vimentin and other type III IF proteins^15,16. Solubility properties of IF proteins are tightly regulated by phosphorylation, a key post-translational modification (PTM) for filament rearrangement and solubility^17,18,19,20. Most IFs undergo extensive regulation by a number of PTMs at conserved sites, resulting in functional changes¹⁷.

The purpose of this method is to introduce investigators who are new to the IF field to biochemical extraction and analytical methods for the study of IF proteins across multiple mouse tissues. Specifically, we focus on isolation of IF proteins using a high-salt extraction method and assessment of changes in PTMs via mass-spectrometry and by PTM-targeting antibodies. These methods build upon previously published procedures²¹ but include modifications for extracting different IF protein types to uncover common mechanism for regulation across the IF family. For example, K8 acetylation at a specific lysine residue regulates filament organization, while hyperacetylation promotes K8 insolubility and aggregate formation²². Recent global proteomic profiling studies have additionally revealed that most tissue-specific IF proteins are also targets for acetylation and that most IF acetylation sites are confined to the highly conserved rod domain. This highlights the need for methods suitable for global profiling of the IF system. We also introduce a rapid method of isolating IF proteins from multiple tissues using automated homogenization in optimized lysing matrix. The resulting preparations are suitable for downstream PTM analysis via mass spectrometry and other methods.

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 M Tris-HCl (pH 7.6), 14 mL of 5 M NaCl, 55.9 g KCl, 5 mL of 0.5 M EDTA, and 2.5 mL of Triton X-100, adjust volume to 500 mL using double distilled water). Store High Salt Buffer solution at 4 °C.
3. Just prior to use, supplement an appropriate amount of Triton-X and High Salt Buffer (e.g. 1 mL for each 25 mg tissue sample) with a protease or protease/phosphatase inhibitor cocktail and discard any unused buffer containing the inhibitors.
4. Prepare 5 mM EDTA in 1x PBS, pH 7.4. To make 500 mL: stir 5 mL of 0.5 M EDTA into 495 mL of 1x PBS, pH 7.4.
5. Isolate different mouse organs (brain, heart, lung, liver, pancreas, colon, intestine, kidney, spleen) using approved protocols that comply with veterinary guidelines and institutional standards²³. The tissue collection procedure should take no more than 5 min to preserve RNA and protein integrity of protease-rich tissues (e.g. pancreas should be processed first).
6. Cut a small amount of tissue (~5-20 mg) and place in RNA storage solution, for subsequent RNA extraction, cDNA synthesis, and quantitative real-time PCR analysis for IF gene expression. Place RNA storage solution tubes with tissue at 4 °C overnight and follow manufacturer protocol for further storage and isolation steps.
7. Cut the rest of the tissue into smaller fragments (e.g. 0.5 cm) and place in cryovial. Snap-freeze and store vials at -80 °C or liquid nitrogen for longer term storage.

2. IF Gene Expression Analysis

1. Extract RNA from tissues preserved in the RNA storage solution reagent. Use any suitable/preferred RNA extraction method according to manufacturer's protocol.
2. Quantify RNA concentration and use 2 μg of RNA to generate cDNA using a suitable reverse transcription kit according to manufacturer's protocol.
3. Using the generated cDNA and mouse IF gene-specific primers set up qPCR reactions, including three technical replicates of each sample as well as blank control according to manufacturer's protocol.
4. Quantify IF gene expression as fold change comparing different conditions (e.g. young versus old tissue).

3. Preparation of Total Tissue Lysates for Immunoblot

1. Homogenize 25 mg of tissue in 1 mL of 2x non-reducing SDS sample buffer. Omit bromophenol blue dye from the sample buffer if a colorimetric protein assay is to be performed to quantify protein concentration.
2. Add 5% (v/v) of 2-mercaptoethanol (2-ME) to make reduced samples. To 200 μL of the non-reducing samples, add 10 μL of 2-ME.
3. Determine protein concentration using detergent- and reducing agent-compatible protein assay. If dye is already included in the sample buffer, the protein amount can be estimated after running a gel via a number of techniques, including Coomassie-based stain²⁴.
4. Vortex all samples and heat at 95 °C for 5 min.
5. Perform western blotting under both reducing and non-reducing conditions. Exposure membrane briefly (<1 min) to reveals monomeric species, and longer (>1 min) to reveal high molecular mass complexes containing IF proteins. If monitoring aggregation, examine entire gel/membrane (including the bottoms of the gel wells).
6. Run a parallel gel and stain with a protein stain as a loading control²⁴. In disease models or injury experiments total protein stain should be used as a loading control, as opposed to immunoblots for 'housekeeping' proteins (e.g. actin, GAPDH) because the latter change under different stress conditions.

4. Preparation of Detergent-soluble and High-salt Extracts of Tissue-specific IFs

1. Add 1 mL of ice-cold Triton X-100 buffer into a glass tube homogenizer and place it on ice.
2. Remove a small piece of tissue (~25 mg) from liquid nitrogen storage and place directly into the glass homogenizer. Use a polytetrafluoroethylene pestle to homogenize (50 strokes) and avoid making bubbles. Keep the homogenizer and lysate cold at all times.
3. Transfer lysate to a 1.5 mL microcentrifuge tube on ice and centrifuge at 20,000 x g for 10 min in a pre-chilled centrifuge (4 °C).
4. Collect the supernatant fraction into a separate tube. This is the Triton X-soluble fraction, which can be used for immunoprecipitation (i.p.) and analysis of the detergent-soluble pool of IF proteins. Note that steps 4.1-4.4 may be repeated to achieve a cleaner IF extract from brain tissue.
5. Add 1 mL of High Salt Buffer to the tissue pellet, transfer to a clean homogenizer and dounce 100 strokes. Transfer the homogenate back to the microcentrifuge tube and place the tube on a rotating shaker in the cold room for 1 h.
6. Centrifuge the homogenates at 20,000 x g for 20 min at 4 °C. Discard the supernatant.
7. Add 1 mL of ice-cold PBS/EDTA buffer to the pellet and homogenize the pellet (20 strokes) in a clean homogenizer as a final clean-up step*. Transfer to a new tube and centrifuge at 20,000 x g for 10 min at 4 °C to obtain the IF protein-rich high salt extract (HSE).
   * Optionally, vortex instead of homogenization at this step.
8. Discard the supernatant and dissolve the pellets in 300 μL of non-reducing SDS sample buffer that has been pre-heated. Break up the pellet initially by pipetting and vortexing, and then heat the samples for 5 min at 95 °C.
9. Vortex and pipet as needed to ensure the pellet is dissolved. It may take several minutes to fully dissolve the pellets.
10. Store all samples at -20 °C until analysis.

5. Automated Tissue Lysis for IF Protein Extraction in High-volume Experiments

1. For RNA extraction, place lysis buffer (600 μL buffer per 25 mg of tissue) in a tube containing lysing matrix D (uses small ceramic spheres) and pulse twice for 25 s in the tissue lyser. Separate lysate from the matrix by centrifugation at 20,000 x g and proceed to the next step in isolation.
2. For protein extraction, place Triton X-100 (or SDS sample buffer if preparing total lysate) in a lysing tube with lysing matrix SS (use a single stainless steel bead). After testing multiple matrices, this was selected because it produces IF protein extracts that are in similar quality as the traditional douncing method. Note that the automated method is not optimal for pancreas and spleen, and the standard homogenization protocol should be used for these tissues.
3. To proceed with preparation of High Salt Extract, remove the stainless steel bead from the tube using a magnet, and centrifuge the tubes at 20,000 x g for 10 min at 4 °C.
4. Continue with step 4.4 (above) of the manual protocol.
5. Store all samples at -20 °C until analysis.

6. Immuno-enrichment of Post-translationally Modified IF Proteins

1. Prepare PBST buffer (0.02% Tween-20 in PBS). To make 50 mL, add 10 μL of Tween-20 to 50 mL of PBS, pH 7.4.
2. Prepare PTM antibody solution (1-10 μg of antibody in 200 μL of PBST). In general, 3 μg of antibody/reaction is a good starting condition that can be further optimized if needed.
3. For each reaction, aliquot 50 μL of magnetic beads into a microcentrifuge tube, place on the magnet and aspirate the bead storage solution.
4. Conjugate the beads to the immunoprecipitation antibody by re-suspending in the antibody solution and incubating on rotator (end-over-end, to ensure mixing of small volumes) at room temperature for 20 min.
5. Place the tubes on magnet and aspirate antibody solution.
6. Rinse the antibody-conjugated beads once in 200 μL PBST and remove wash buffer.
7. Add 0.6-1 mL of the tissue lysate to the beads, mix by gentle pipetting and incubate for 3 h on rotator in a cold room.
8. Place tubes on magnet, remove lysate, and wash the beads five times with 200 μL of PBST. After the last washing step, collect the beads in 100 μL of PBS (no Tween-20) and transfer to a clean new tube. Place the tube on the magnet.
9. Aspirate the PBS and add 100 μL of non-reducing sample buffer. Remove 50 μL and add 2-ME (5%) to make reducing samples. Heat the samples to 95 °C for 5 min.
10. Separate the i.p. fraction from the beads on the magnet and collect it into a new tube.
11. Store samples at -20 °C until analysis.

7. Preparation of IF Protein Samples for Mass Spectrometry Analysis

1. Schedule a consultation with a proteomics expert prior to initiating a study since there is significant time and cost involved with mass spectrometry analysis.
2. Take special precautions to avoid contamination. Handle all gels with clean gloves and incubate in clean containers, washed only using ddH₂O (avoid soap).
3. Run 20-50 μL of the HSE sample (from Sections 4 and 5) on an SDS-PAGE gel according to standard conditions.
4. Stain with a protein stain for 1 h. Rinse multiple times and de-stain in ddH₂O overnight. The IF protein bands should be easily visible after de-staining.
5. Place the gel between plastic sheet protectors, scan and mark the bands that will be excised and sent for analysis.
6. Excise the IF protein bands using a new clean razor.
7. Place the gel bands in clean microcentrifuge tubes and transfer to a mass spectrometry facility.

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