Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

Summary

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

Abstract

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer's disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

Introduction

Tau was originally identified as a heat-stable microtubule-associated protein that was co-purified with tubulin¹. Tau is exclusively expressed in higher eukaryotes^2,3,4. The main function of tau is to control microtubule assembly^1,5,6. It also contributes to polymerization of microtubules⁷, axonal transport⁸, changes in axonal diameter⁹, formation of neuroma polarity, and neurodegeneration¹⁰. Tau also acts as a protein scaffold to control some signaling pathways. Rat brain studies suggest that tau is neuron-specific and that it primarily localizes in axons¹¹. Because tau is essential for microtubule polymerization and neuronal development, tau was hypothesized to play a major role in axonal development in the central nervous system; this hypothesis was later verified by in vitro and in vivo experiments. In addition to neurons, tau is expressed in different non-neuronal cells, including liver, kidney, and muscle cells^12,13. Tau is expressed also in human breast, prostate, colorectal, gastric, and pancreatic cancer cell lines and tissues^{14,15,16,17,18,19}. Tau is also found in inclusion-body myositis as twisted tubulofilaments in inclusion bodies²⁰.

Tau may carry several post-translational modifications. Of all post-translational modifications, phosphorylation is the most common. Increased tau phosphorylation decreases its affinity for microtubules, finally destabilizing the cytoskeleton. Eighty-five phosphorylation sites have been described in tau protein isolated from human Alzheimer's disease brain tissues. Of these sites, 53% constitute serine, 41% threonine, and only 6% tyrosine residues^21,22,23. Tau phosphorylation affects its localization, function, binding, solubility, and its susceptibility to other post-translational modifications. Also tau phosphorylation to more than the normal extent (or fully saturated with phosphate groups) is known as hyperphosphorylation that replicates structural and functional characteristics of Alzheimer's disease²⁴. Tau maintains proper functioning of the axonal microtubules and ensures normal neuronal functioning under physiological conditions. However, hyperphosphorylated tau fails to maintain a well-organized microtubule binding, causing neuronal loss because of microtubule disassembly. Normal levels of tau phosphorylation are required for proper tau functioning, but tau fails to function normally if its characteristic phosphorylation level is altered and if it is hyperphosphorylated²⁵. In Alzheimer's disease and some other age-related neurodegenerative disorders, tau becomes hyperphosphorylated and forms the paired helical filaments and neurofibrillary tangles^26,27. Thus, methods for determining tau phosphorylation and microtubule binding are important.

Colorectal cancer, an ageing-associated cancer, is the third most frequently diagnosed cancer and the third prominent death-causing cancer for both men and women²⁸. Colorectal cancer is one of the main death-causing cancers in the Western world²⁹. Because both colorectal cancer and Alzheimer's disease are associated with ageing and both happen mainly in the developed countries where people enjoy similar dietary habits, the two diseases may somehow be correlated. In addition, tau-positive and tau-negative cancer cells respond differently to chemotherapeutic agents, e.g., paclitaxel¹⁶.

Curcumin is one of the main derivatives of Curcuma longa, the Indian spice turmeric³⁰. For centuries, South Asian populations have consumed turmeric in their diets on a daily basis. Curcumin is used to treat different diseases, including colorectal cancer, Alzheimer's disease, diabetes, cystic fibrosis, inflammatory bowel disease, arthritis, hyperlipidemia, atherosclerosis, and ischemic heart disease^{31,32,33,34,35,36,37,38}. Lithium can also kill colorectal cancer cells or prevent their proliferation³⁹. Lithium can also be used for treating Alzheimer's disease⁴⁰ as it decreases tau aggregation and prevents its hyperphosphorylation as observed in a transgenic mouse model^41,42,43,44.

This manuscript aims to: 1) measure the total tau and phospho-tau expression levels in treated cells; 2) describe a phosphatase assay to measure overall tau phosphorylation; 3) examine microtubule-binding of tau; and 4) localize tau by confocal microscopy in colorectal cancer cell lines treated with curcumin or LiCl. Results reveal that cell treatment with curcumin, which is a supposedly good chemotherapeutic agent for colon cancer, and treatment with LiCl can reduce expression of both total tau and phosphorylated tau in colorectal cancer cell lines. These treatments can also cause nuclear translocation of tau. However, unexpectedly, curcumin fails to improve binding of tau to microtubules.

Protocol

1. Preparation of Reagents

1. Add 10 µL of (1 mM) phenylmethylsulfonyl fluoride solution, 10 µL (1x from 100x stock) of protease inhibitor cocktail solution, and 10 µL (1x from 100x stock) of phosphatase inhibitor cocktail solution to 1 mL of 1x radioimmunoprecipitation assay (RIPA) buffer to prepare the complete RIPA lysis buffer.
2. Prepare 10x general tubulin buffer (PEM) buffer.
   1. Add 800 mM (6.0474 g) piperazine-N-N'-bis-2-ethanesulfonic acid, 10 mM (95.1 mg) ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 mM (51 mg) MgCl₂, and 1.25 M (3 mL of 10 M) NaOH in a 50-mL tube, mix with 15 mL distilled water, and dissolve by using a vortex. Check the pH (should be 6.9). If pH > 6.9, discard the buffer and remake a fresh lot.
      NOTE: NaOH should be below 1.25 M to ensure that pH does not overshoot 6.9. It is not advised to use HCl for adjusting pH >6.9.
   2. Add NaOH dropwise until pH is 6.9. Add distilled water to make up to 25 mL of 10X PEM buffer. Finally, filter using a syringe and a 0.2 µm syringe filter. Divide this buffer into aliquots in 1.5 mL tubes and store at 4 °C.
3. Prepare 5x sample buffer with reducing agent and dye.
   1. Mix 300 µL (60 mM) of 1 M Tris-HCl (pH 6.8), 2.5 mL (25%) of 50% glycerol, 1 mL (2%) of 10% sodium dodecyl sulfate (SDS), and 1.2 mL distilled water to make up 5 mL of 5x SDS sample buffer. Store at −20 °C.
      NOTE: Because glycerol is viscous, measuring 1.25 mL glycerol is difficult. One mL of 100% glycerol weighs 1.26 g. Thus, weigh 1.575 g of 100% glycerol (which equals 1.25 mL of 100% or 2.5 mL of 50% glycerol) in a 15 mL tube first; mix well with the other components.

2. Cell Culture, Treatment with Curcumin or LiCl Treatment, and Examination of Protein Expression

1. Add ~1 x 10⁶ HCT 116 cells into 100 mm tissue-culture dishes containing the Minimum Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After one day of culturing, cells will reach 60-70% confluence.
   NOTE: The number of plates needed depends on the number of treatments described below.
2. When cells reach ~65% confluence (approximated using microscopy), remove the MEM supplemented medium and add serum-free MEM with 5 µM, 10 µM, and 30 µM curcumin or 25 mM, 50 mM, and 100 mM LiCl. Incubate at 37 °C for 24 h in a humidified atmosphere containing 5% CO₂.
3. 24 h after treatment, wash the cells with 1 mL ice-cold phosphate-buffered saline (PBS), and scrape the cells using a cell scraper. Transfer the scraped cells into 1.5 mL centrifuge tubes and centrifuge for 4 min at 1,800 x g at 4 °C to obtain cell pellets.
4. Lyse the cells by suspending the pellets in 100 µL of complete RIPA buffer at 4 °C for 20 min while regularly tapping the tubes. Sonicate the samples briefly.
5. Centrifuge the cell homogenates in a benchtop centrifuge at 22,570 x g for 20 min at 4 °C. Transfer the supernatants to other labeled tubes using a pipette.
6. Measure the protein concentration in the supernatants by the Bradford assay⁴⁵ using commercial protein-assay kits.
7. Prepare samples of equal protein concentration (10 µg protein/13 µL sample) of cell lysates with 4X SDS gel-loading buffer. Prepared 4x SDS gel-loading buffer fresh containing 400 mM dithiothreitol (DTT).
   NOTE: At this stage, samples can be stored at −20 °C if needed.
8. Incubate the samples on a heat block at 100 °C for 5 min. Mix the samples by using a vortex and wait to cool them for ~10-15 min.
9. Assemble an electrophoresis system.
   1. Load 10 µg protein (13 µL sample) per well on a 10% SDS-polyacrylamide gel for electrophoresis (SDS-PAGE). Load the molecular-weight ladder onto the gel to one lane.
      NOTE: As electrophoresis starts, the protein ladder will resolve into protein bands of apparent molecular weights. Use these bands to estimate the molecular weights of the unknown protein bands.
   2. After loading the samples, connect the positive and negative electrodes of the electrophoresis system to a power source to maintain a constant voltage. Initially, start at 70 V for 20 min to move the protein samples from the stacking gel into the resolving gel. Then, increase the voltage to 125 V for approximately 70-120 min until samples and protein ladder reach the end of the gel.
   3. After completion of electrophoresis, transfer the gel to a polyvinylidene difluoride (PVDF) membrane using a wet electroblotting system. Transfer for ~100 min at 100 V. Block the membrane in 4% bovine serum albumin (BSA) blocking buffer for 90 min at room temperature of around 25 °C.
10. Mark the blot by cutting it on the side with the molecular-weight ladder. Use a transparent plastic sheet and a sharp cutter for cutting the blot. Prepare the primary-antibody solutions (anti-tau at 1:5,000; anti-phospho-tau at 1:4,000) and incubate the blot in this solution at 4 °C overnight.
11. After overnight incubation, wash the blot for 7 min four times in phosphate-buffered saline-Tween-20 (PBST) solution.
12. Prepare the relevant secondary-antibody solutions (goat anti-rabbit or anti-mouse IgG at 1:5,000), and incubate the blot in this solution for 90 min at room temperature of around 25 °C. Wash in PBST four times, 7 min each.
13. Develop the blot using a chemiluminescence kit according to the manufacturer's recommendations. Cover the blot using a transparent plastic wrap. Acquire images by a chemiluminescence imaging system for chemiluminescence with relevant software.

3. Phosphatase Assay

1. Treat the cell lysates (from step 2.5) with alkaline phosphatase in the alkaline phosphatase buffer.
   1. For both control and treated samples, prepare 20 µL samples containing 20 µg total protein. Add the volume of cell lysates that contains 20 µg total protein, followed by 2 µL alkaline phosphatase buffer and 10 µL alkaline phosphatase. Add distilled water to make up 20 µL.
2. Incubate the samples at 37 °C for 1 h. Stop the reaction by adding ethylenediaminetetraacetic acid (EDTA) at a final concentration of 50 mM, or by adding sodium orthovanadate (Na₃VO₄) at a final concentration of 10 mM. The reaction can also be stopped by heating at 75 °C for 5 min.
3. Before the incubation of samples with phosphatase finishes, prepare samples of the same concentration without adding phosphatase for comparison with phosphatase-treated samples.
4. Add the freshly prepared 4X SDS gel-loading buffer containing 400 mM DTT.
5. Incubate the samples on a heat block at 100 °C for 5 min. Mix the samples using a vortex. Cool at room temperature for ~ 10-15 min.
6. Assemble an electrophoresis system for running a 10% polyacrylamide gel by SDS-PAGE.
   1. Load 10 µg protein (13 µL sample) per well on a 10% polyacrylamide gel. Load the molecular-weight ladder.
   2. After loading the sample, connect the positive and negative electrodes of the electrophoresis system to a power source to maintain a constant voltage. Initially, start at 70 V for 20 min to move the protein samples from the stacking gel into the resolving gel. Then, increase the voltage to 125 V and run the gel for approximately 70-120 min until samples and protein ladder reach the end of the gel.
   3. After completion of electrophoresis, transfer the gel onto a PVDF membrane using a wet electroblotting system. Transfer for ~100 min at 100 V.
   4. Mark the blot by making a small cut on the side the molecular-weight ladder. Use a transparent plastic sheet and a sharp cutter for cutting the blot.
7. Block the membrane in 4% BSA blocking buffer for 90 min at room temperature.
8. Incubate the blot in primary-antibody solution (anti-tau at 1:5,000) at 4 °C overnight. Wash the blot in PBST four times, for 7 min each.
9. Prepare the relevant secondary-antibody solution (goat anti-mouse IgG at 1:5,000), and incubate the blot for 90 min at room temperature. Wash in PBST four times, for 7 min each.
10. Develop the blot using a chemiluminescence kit according to the manufacturer's recommendations. Cover the blot within a transparent plastic wrap. Acquire images by a chemiluminescence imaging system for chemiluminescence with relevant software.

4. Microtubule-binding Assay

1. For the microtubule binding assay, repeat steps 2.1-2.6.
2. Prepare 1X (2 mL) PEM buffer from 10X PEM buffer stored at 4 °C by adding 20 µM (40 µL) paclitaxel and 1 mM (20 µL) GTP. Take the microtubule (MT) stock from −80 °C freezer and the E. coli tau working stock from −20 °C freezer.
3. Prepare samples as per Table 1.
4. Incubate at 37 °C for 30 min. Ultracentrifuge at 25 °C for 60 min at 100,000 x g.
5. Transfer 120 µL of supernatants containing unbound tau, to clean and labeled tubes.
6. Add 120 µL (same volume as supernatant) of 5X sample buffer to the pellet containing bound tau and mix thoroughly. Transfer the solution to clean labeled tubes and mix thoroughly.
7. Measure the protein concentration (see step 2.6).
   NOTE: While preparing samples, use the volume of solution (step 4.6) for preparing both the pellet and supernatant samples.
8. Prepare samples of equivalent protein concentration and add freshly prepared 4x SDS gel-loading buffer containing DTT (400 mM).
   NOTE: Samples can be stored at −20 °C at this stage.
9. Repeat steps 3.5-3.10.

Sample	Name		Microtubule needed		Main Protein needed		PEM-GTP-PTX
1	HCT 116-Control (6.21 μg/μl)		2 μl		9.66 μl (60 μg)		108.34 μl
2	HCT 116-Curcumin 10 μM (4.81 μg/μl)		2 μl		16.63 μl (80 μg )		101.37 μl
3	HCT 116-Curcumin 20 μM (3.28 μg/μl)		2 μl		30.49 μl (100 μg)		87.51 μl
4	HCT 116-LiCl 25 mM (5.43 μg/μl)		2 μl		18.42 μl (100 μg)		99.58 μl
5	E. coli tau		2 μl		1 μl tau-352		117 μl
6	MT Only		2 μl		X		118 μl
							120 μl each

Table 1: Sample preparation for microtubule-binding assay.

5. Localization and Expression of Tau after Treatment of Cells with Curcumin

1. Clean a coverslip using 70% ethanol and dry it using a laboratory tissue. Insert a single coverslip in each well of a labeled 6-well tissue-culture plate. Place the plate on a biological safety cabinet and switch on a UV light for at least 3 h.
2. Subculture the cells and seed them into the relevant wells of the 6-well plate at 2.5 x 10⁵ cells/well in recommended culture medium.
3. After ~24 h, examine the cells using an inverted microscope by using both the 10X and 40X objectives to check for cellular morphological changes. Record photos if needed.
4. Rinse the cells twice using PBS. Fix the cells in 3.7% formaldehyde in a 37 °C incubator in the dark.
   NOTE: Fixation by formaldehyde at room temperature works well, too. Wear appropriate personal protective equipment when handling formaldehyde.
5. Wash the cells in PBS. Fix cells in ice-cold methanol at −20 °C for 15 min. Permeabilize the cells by incubating them in 3% BSA and 0.1% Triton X-100 in PBS on ice for 10 min. Wash the cells with PBS.
6. Incubate the cells in blocking buffer (3% BSA and 0.1% Tween-20 in PBS solution) for 1 h at room temperature.
   NOTE: Incubation for 2 h at 4 °C works well, too.
7. Prepare the primary-antibody (anti-tau at 1:200) solution in 3% BSA and 0.1% Tween-20 in PBS (e.g., 2 µL tau-13 antibody in 0.4 mL buffer).
8. Cut small pieces of a PVDF membrane so that the pieces fit properly within each well of the 6-well plate; soak them in water for 5 min. Place the cut PVDF in each well.
9. Add 60 µL of primary antibody solution to the cut PVDF pieces in each well. Place the coverslips such that the cells can touch the primary-antibody solution. Incubate at 4 °C in a dark, humid chamber overnight. Wash in PBS four times.
10. Prepare the secondary-antibody solution (fluorescein-conjugated 594 anti-mouse IgG at 1:400) in 3% BSA and 0.1% Tween-20 in PBS.
11. Cut small pieces of a PVDF membrane so that the pieces fit properly within each well of the 6-well plate, and soak them in water for 5 min. Place the cut PVDF in each well.
12. Add 80 µL of the secondary-antibody solution to each of the PVDF pieces in the 6-well plate. Place the coverslip such that the cells can touch the secondary-antibody solution. Incubate in a dark, humid chamber at room temperature for 2 h. Wash with PBS four times.
13. Mount samples in the mounting medium with 4',6-diamidino-2-phenylindole (DAPI) and fix the coverslips on glass slides.
    1. Add one drop of the mounting medium with DAPI on glass slides. Remove the coverslips from each well and place them on the glass slides containing the mounting medium with DAPI. Ensure that the surface of each coverslip containing stained cells touches the mounting medium on the corresponding glass slide.
    2. Apply nail polish all around each coverslip to seal and fix them airtight on the glass slides. If needed, do this step twice.
14. Incubate for 1.5 h at room temperature in a dark chamber.
15. Examine the slides using a confocal microscope by using immersion oil on the coverslip in a dark room.

Results

Expression of total tau and phospho-tau was examined after treating the cells with different concentrations of curcumin or LiCl (Figure 1). Treatment of cells with the three different concentrations of curcumin decreased tau expression levels; however, phospho-tau expression increased upon treatment with low concentration of curcumin but decreased upon treating cells with higher curcumin concentrations. Anti-phospho-tau (Ser396) was used for detection of phos...

Discussion

This manuscript established different procedural conditions for detecting total tau and phosphorylated tau in colorectal cancer cells treated with curcumin and LiCl. To assess the overall phosphorylation status of tau in protein samples, a phosphatase assay was described. This assay can potentially be used to examine the phosphorylation status of any protein.

This assay is based on the principle that phosphorylated protein moves slower than its non-phosphorylated state. Alkaline phosphatase an...

Materials

Name	Company	Catalog Number	Comments
HCT 116 cell	ATCC	CCL-247
MEM (EBSS)	Hyclone	SH30024.01
Fetal Bovine Serum (FBS)	ThermoFisher (Gibco)	16000044	Store at -20 °C
penicillin-streptomycin	Hyclone	SV30010
Trypsin-EDTA solution	WelGene	LS 015-01
100 mm dish	Corning	430161
6 well plate	Corning	Coster 3516
Anti-Tau 13 antibody	abcam	ab19030
Dithiothreitol (DTT)	Roche	10 708 984 001	Storage Temperature 2–8 °C
Microlitre Centrifuges	Hettich Zentrifugen	MIKRO 200 R
Paclitaxel	Sigma-Aldrich	T1912	Storage Temperature 2–8 °C
Curcumin	Sigma-Aldrich (Fluka)	78246	Storage Temperature 2–8 °C
Microtubules (MT)	Cytoskeleton	MT001	Store at 4 °C (desiccated)
Mounting Medium with DAPI	Vector Laboratories	H-1200	Store at 4 °C in the dark
Sodium hydroxide	Sigma	72068
Magnesium Chloride	Sigma-Aldrich	M2670
GTP	Sigma-Aldrich	G8877	Store at -20 °C
DPBS	WelGene	LB 001-02
Sonic Dismembrator	Fisher Scientific	Model 500
Ultracentrifuge	Beckman Coulter	Optima L-100 XP
PIPES	Sigma	P1851
Bovine serum Albumin (BSA)	Sigma	A7906
Molecular Imager	Bio-Rad	ChemiDoc XRS+	Store at 4 °C
Protein assay dye reagent	Bio-Rad	500-0006
α-tubulin (11H10) Rabbit mAb	Cell signalling	2125
GAPDH (14C10) Rabbit mAb	Cell signalling	2118
Anti-Tau (phospho S396) antibody	abcam	ab109390
EGTA	Sigma	E3889	Store at room temperature
FastAP Thermosensitive Alkaline Phosphatase	Thermo Scientific	EF0651	Store at -20 °C
PMSF	Sigma	P7626	Store at room temperature
Phosphatase Inhibitor Cocktail	Cell Signalling	5870	Store at 4 °C
Protease Inhibitor Cocktail	Cell Signalling	5871	Store at 4 °C
RIPA Buffer	Sigma	R 0278	Storage Temperature 2–8 °C
Tau-352 human	Sigma	T 9950	Store at -20 °C
Triton X-100	Sigma-Aldrich	X - 100	Store at around 25 °C
PVDF membrane	Bio-Rad	162-0177
Goat anti-mouse IgG Secondary Antibody	ThermoFisher	A-11005	Store at 4 °C in the dark
Confocal Microscopy	Leica Microsystem	Leica TCS SP5
Sodium Dodecyl Sulfate (SDS)	Affymetrix	75819
Protein Assay	Bio-Rad	500-0006	Store at 4 °C