Name: assay for phosphorylation and microtubule binding along with localization of tau protein in colorectal cancer cells
Uploaded: 2017-10-10T12:30:00
Description: Watch this Scientific Journal Video about Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells at JoVE.com

This research was performed as part of the project titled &#39;Development and industrialization of high value cosmetic raw materials from marine microalgae&#39;, funded by the Ministry of Oceans and Fisheries, Korea, and was supported by an intramural grant (2Z04930) from KIST Gangneung Institute of Natural Products.

1. Preparation of Reagents
<ol>
	<li>Add 10 &#181;L of (1 mM) phenylmethylsulfonyl fluoride solution, 10 &#181;L (1x from 100x stock) of protease inhibitor cocktail solution, and 10 &#181;L (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.</li>
	<li>Prepare 10x general tubulin buffer (PEM) buffer.
	<ol>
		<li>Add 800 mM (6.0474 g) piperazine-N-N&#39;-bis-2-ethanesulfonic acid, 10 mM (95.1...

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...

Tau was originally identified as a heat-stable microtubule-associated protein that was co-purified with tubulin1. Tau is exclusively expressed in higher eukaryotes2,3,4. The main function of tau is to control microtubule assembly1,5,6. 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 cells12,13. Tau is expressed also in human breast, prostate, colorectal, gastric, and pancreatic cancer cell lines and tissues14,15,16,17,18,19. 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&#39;s disease brain tissues. Of these sites, 53% constitute serine, 41% threonine, and only 6% tyrosine residues21,22,23. 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&#39;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&#39;s disease and some other age-related neurodegenerative disorders, tau becomes hyperphosphorylated and forms the paired helical filaments and neurofibrillary tangles26,27. 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&#39;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&#39;s disease, diabetes, cystic fibrosis, inflammatory bowel disease, arthritis, hyperlipidemia, atherosclerosis, and ischemic heart disease31,32,33,34,35,36,37,38. Lithium can also kill colorectal cancer cells or prevent their proliferation39. Lithium can also be used for treating Alzheimer&#39;s disease40 as it decreases tau aggregation and prevents its hyperphosphorylation as observed in a transgenic mouse model41,42,43,44.
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.

<table>
	<tbody>
		<tr>
			<td>HCT 116 cell</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (EBSS)</td>
			<td>Hyclone</td>
			<td>SH30024.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher (Gibco)</td>
			<td>16000044</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>WelGene</td>
			<td>LS 015-01</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm dish</td>
			<td>Corning</td>
			<td>430161</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>Coster 3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tau 13 antibody</td>
			<td>abcam</td>
			<td>ab19030</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Roche</td>
			<td>10 708 984 001</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Microlitre Centrifuges</td>
			<td>Hettich Zentrifugen</td>
			<td>MIKRO 200 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Sigma-Aldrich</td>
			<td>T1912</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Curcumin</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>78246</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Microtubules (MT)</td>
			<td>Cytoskeleton</td>
			<td>MT001</td>
			<td>Store at 4 &#176;C (desiccated)</td>
		</tr>
		<tr>
			<td>Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Store at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>WelGene</td>
			<td>LB 001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>Model 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima L-100 XP</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDoc XRS+</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Protein assay dye reagent</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin (11H10) Rabbit mAb</td>
			<td>Cell signalling</td>
			<td>2125</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH (14C10) Rabbit mAb</td>
			<td>Cell signalling</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tau (phospho S396) antibody</td>
			<td>abcam</td>
			<td>ab109390</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>FastAP Thermosensitive Alkaline Phosphatase</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail</td>
			<td>Cell Signalling</td>
			<td>5870</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Cell Signalling</td>
			<td>5871</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Sigma</td>
			<td>R 0278</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Tau-352 human</td>
			<td>Sigma</td>
			<td>T 9950</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>X - 100</td>
			<td>Store at around 25 &#176;C</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Bio-Rad</td>
			<td>162-0177</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>A-11005</td>
			<td>Store at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Confocal Microscopy</td>
			<td>Leica Microsystem</td>
			<td>Leica TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Affymetrix</td>
			<td>75819</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Assay</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td>Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

assay for phosphorylation and microtubule binding along with localization of tau protein in colorectal cancer cells

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&#39;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.

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...

Watch this Scientific Journal Video about Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells at JoVE.com

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

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.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

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