We thank Shigeo Okabe and Hitomi Matsuno for the low-density culture of hippocampal neurons, Masayoshi Mishina for TLCN-deficient mice, Sachiko Mitsui and Momoko Shiozaki for technical assistance, and members of the Yoshihara laboratory for helpful discussions. This work was supported by JSPS KAKENHI Grant Nos. JP20700307, JP22700354, and JP24500392 and MEXT KAKENHI Grant Nos. JP23123525 to YF and JP20022046, JP18H04683, and JP18H05146 to YY.

All methods described here have been approved by the Institutional Animal Care and Use Committee of RIKEN Wako.
1. Culture of Hippocampal Neurons
<ol>
	<li>Preparation of culture medium
	<ol>
		<li>Preparation of 200x Vitamin mix. Dissolve 100 mg of D-pantothenic acid hemicalcium salt, 100 mg of choline chloride, 100 mg of folic acid, 180 mg of i-inositol, 100 mg of niacinamide, 100 mg of pyridoxal HCl, and 100 mg of thiamine HCl in 500 mL of ultrapure water using a magnetic stirrer. The solution is not completely dissolved. Carefully mix, aliquot in 50 mL tubes and store at -20 &#176;C.</li>
		<li>Preparation of Riboflavin solution. Dissolve 100 mg of Riboflavin in 500 mL of ultrapure water using a magnetic stirrer. The solution is not completely dissolved. Carefully mix, aliquot in 50 mL tubes and store at -20 &#176;C.</li>
		<li>Preparation of 1 M CaCl2. Dissolve 7.35 g of CaCl2&#183;2H2O in 50 mL of ultrapure water using a magnetic stirrer.</li>
		<li>Preparation of Minimum essential medium (MEM). Dissolve 400 mg of KCl, 6800 mg of NaCl, 2,200 mg of NaHCO3, 158 mg of NaH2PO4&#183;2H2O, 7000 mg of D-glucose, and 200 mg of MgSO4-7H2O in 950 mL of ultrapure water using a magnetic stirrer.</li>
		<li>Titrate 1.8 mL of 1 M CaCl2 to the MEM in a drop-by-drop manner using a 1 mL pipet with a constant agitation on a magnetic stirrer. Adjust the pH of the MEM to pH 7.25 with 1 mol/L HCl.</li>
		<li>Add 5 mL of 200x Vitamin mix and 200 &#956;L of Riboflavin solution to the MEM. Adjust the volume of the solution to 1,000 mL with ultrapure water. Filter the solution using a 0.22 &#956;m filter system and store it at 4 &#176;C.</li>
		<li>Preparation of 10x DNase-I stock solutions. Dissolve 100 mg of DNase-I in 12.5 mL of Hanks&#39; Balanced Salt Solution (HBSS), filter through a 0.22 &#956;m filter, aliquot in 1.5 mL tubes, and store the tubes at -20 &#176;C.</li>
		<li>Preparation of cytosine &#946;-D-arabinofuranoside (Ara-C) stock solution. Dissolve 25 mg of Ara-C in 8.93 mL of ultrapure water (final concentration of 10 mM), filter through a 0.22 &#956;m filter, aliquot in 1.5 mL tubes and store at -20 &#176;C</li>
		<li>Preparation of Plating medium. Mix 1 mL of MEM amino acid solution, 750 &#956;L of 1 M HEPES, 1 mL of B27, 125 &#956;L of 200 mM glutamine, 250 &#956;L of penicillin/streptomycin, 2.5 mL of fetal bovine serum (FBS), and 44.375 mL of MEM in a 50 mL tube.</li>
		<li>Preparation of Stop medium. Mix 1 mL of MEM amino acid solution, 750 &#956;L of 1 M HEPES, 5 mL of FBS (final 10%), and 43.25 mL of MEM in a 50 mL tube.</li>
	</ol>
	</li>
	<li>Preparation of poly-L-Lysine-coated dishes
	<ol>
		<li>Coat 35 mm plastic cell culture dishes with 0.2 mg/mL of poly-L-lysine hydrobromide for 1 day at 25 &#176;C. 
		NOTE: Poly-L-lysine should not be used instead of poly-L-lysine hydrobromide.</li>
		<li>Wash the dishes with 2 mL of ultrapure water 3 times. Incubate the dishes with 1.5 mL of Stop medium at 25 &#176;C until use.</li>
	</ol>
	</li>
	<li>Dissection of hippocampal neurons from mouse embryo
	<ol>
		<li>Tissue source of hippocampal neurons. Dissect the hippocampus from wild-type and TLCN-deficient C57BL6/J mice on the embryonic days 16-17 according to the method of Lu et al.19.</li>
		<li>Incubate dissected hippocampi in 0.25% trypsin and 1x DNaseI in HBSS containing 15 mM HEPES, pH 7.2 for 15 min at 37 &#176;C with agitation every 3 min. Remove the solution. Incubate the hippocampi in 10 mL of STOP medium to inactivate trypsin at 4 &#176;C for 5 min.</li>
		<li>Move the hippocampi into 10 mL of fresh STOP medium and incubate at 4 &#176;C for 5 min. Move the hippocampi into 10 mL of fresh STOP medium and incubate at 4 &#176;C for another 5 min. Move the hippocampi into 900 &#956;L of STOP medium and 100 &#956;L of 10x DNaseI in a 15 mL tube. Dissociate the hippocampi into isolated neurons by pipetting 20 times using a 1 mL pipet. 
		NOTE: The tip of the 1 mL pipet slightly touches the bottom of the 15 mL tube during dissociation of the hippocampi.</li>
		<li>Add 9 mL of plating medium, and filter through a 70 &#956;m cell strainer into a 50 mL tube. Count the number of cells using a hemocytometer and adjust to 3.5 x 104 cells/mL in plating medium.</li>
		<li>Aspirate STOP medium from poly-L-lysine-coated dishes. Plate the cells on poly-L-Lysine-coated dishes at 7 x 104 cells/dish (2 mL/dish).</li>
		<li>After 60-64 h of incubation under 5% CO2 at 37 &#176;C, add 2 &#956;L of Ara-C stock solution (final 10 &#956;M) to the neurons, and shake the dish slowly. Keep the culture dishes in a humidified box without changing the culture medium under 5% CO2 at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Purification of Dendritic Filopodia-rich Fraction
<ol>
	<li>After 13 days in vitro (DIV), add magnetic polystyrene microbeads (3 x 106 particles/dish) to 20 dishes containing the cultured neurons. After 1 day, wash the neurons in 1 mL of PBS with agitation 3 times to remove the medium and unbound microbeads. After removing PBS, lyse the neurons with 500 &#956;L/dish of lysis buffer (PBS containing 0.01% Triton X-100, EDTA-free protease inhibitor cocktail, and phosphatase inhibitor cocktail).</li>
	<li>Collect the lysate with a cell scraper and move the lysate to low protein-binding microtubes (10 tubes). Set the tubes on a magnet separator and wait for 1 min. Collect the supernatant and use it as the unbound fraction for silver staining and Western blot analysis.</li>
	<li>Transfer the beads to a new low-protein-binding microtube, set on a magnetic separator, and completely remove the supernatant. Add 500 &#956;L of lysis buffer and wash the beads using a vortex mixer for 15 s. Set the tube on a magnet separator, wait for 1 min, remove the supernatant, and add 500 &#956;L of lysis buffer. Repeat the washing of the beads 10 times and remove the supernatant.</li>
	<li>Elute proteins bound to the beads (the bound fraction) by the addition of 50 &#956;L of 1x SDS sample buffer (62.5 mM Tris HCl, pH 6.8, 2.5% SDS, and 10% glycerol) and boil the tube at 98 &#176;C for 5 min. Centrifuge the tube at 860&#160;x g for 10 s and set the tube on a magnetic separator for 1 min. Collect the supernatant and use it as the bound fraction. 
	NOTE: The protocol can be paused here.</li>
	<li>Measure protein concentrations of the unbound and bound fractions by the BCA protein assay. Visualize protein solutions with bromophenol blue and adjust the concentration to 5 ng/&#956;L for SDS-PAGE.</li>
</ol>
3. Silver Staining and Western Blot Analysis
<ol>
	<li>Separate the bound and unbound fractions (50 ng) by SDS-PAGE using a 5-20% gradient gel. Silver-stain the gel.</li>
	<li>Western blot using anti-TLCN-C (1/3,000), anti-bovine vitronectin (1/5,000), anti-actin (1/1,000), and anti-&#945;-tubulin (1/1,000) as primary antibodies and HRP-conjugated goat anti-rabbit IgG (1/5,000) as the secondary antibody. Visualize the antigens using chemiluminescent Western Blotting Detection Reagent and a chemiluminescence imager.</li>
</ol>

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The authors have nothing to disclose.

We developed a purification method for the dendritic filopodia-rich fraction using affinity between the cell adhesion molecule TLCN and the extracellular matrix protein vitronectin. Compared to PSD fraction, it could be possible to identify the synaptic proteins acting on the immature synapse from the dendritic filopodia-rich fraction. Thus, the constituents of the dendritic filopodia-rich fraction are different from those of the PSD fraction by 74%. Different from PSD fraction, we used cultured hippocampal neurons to ac...

Dendritic filopodia are thought to be precursors of spines. Actin filaments in the dendritic filopodia regulate their extension and retraction1,2,3. After contacting with an axon, selected dendritic filopodia begin their maturation into spines, and a synapse is formed4,5. Components of spines have been determined from comprehensive analysis of postsynaptic density fractions6,7, while components of dendritic filopodia remain largely unknown. It has been shown that telencephalin (TLCN), ERM, SynGAP, Ras, PI3 kinase, Akt, mTOR, polo-like kinase 2, CaMKII, syndecan-2, paralemin-1, ARF6, and EphB regulate dendritic filopodia formation5,8,9,10,11, while a method has not been developed for the comprehensive analysis of molecules present in the dendritic filopodia.
TLCN (ICAM-5) is specifically expressed by spiny neurons in the most rostral brain segment, the telencephalon12. TLCN has 9 Ig-like domains in its extracellular region, a transmembrane region, and a cytoplasmic tail13. TLCN binds to vitronectin (VN) and LFA-1 integrin in its extracellular region, to presenilin in its transmembrane region, and to phospho-ERM and &#945;-actinin in its cytoplasmic region5,8,14,15,16. TLCN binds to the actin cytoskeleton through phospho-ERM at the tips of dendritic filopodia and &#945;-actinin in spines and dendritic shafts8,16.
We showed that overexpression of TLCN enhanced dendritic filopodia formation and induced the reversion of spines to filopodia10. The constitutive active form of ezrin bound to the TLCN cytoplasmic region and enhanced dendritic filopodia formation8. Thus, TLCN regulates dendritic filopodia formation through actin-binding proteins. Esselens et al. demonstrated that microbeads induced TLCN accumulation on cultured neurons17. We showed that phagocytic cup structures were formed on neuronal dendrites around VN-coated microbeads in a TLCN-dependent manner15. Constituents of dendritic filopodia are similar to those of the phagocytic cup. It is difficult to collect dendritic filopodia, but it is relatively easier to collect the phagocytic cup using magnetic microbeads. Thus, we developed a method to purify the phagocytic cup instead of dendritic filopodia18. Here, we describe the purification method for the dendritic filopodia-rich fraction.

<table><tbody><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>1.7 mLl Low Binding MCT</td><td>Sorenson BioScience</td><td>39640T</td><td></td></tr><tr><td>200 mM L-Glutamine</td><td>Gibco</td><td>2530149</td><td></td></tr><tr><td>35 mm plastic cell culture dishes</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>Anti-actin</td><td>Sigma-Aldrich</td><td>A-5060</td><td></td></tr><tr><td>Anti-alpha-Actinin</td><td>Sigma-Aldrich</td><td>A-5044</td><td></td></tr><tr><td>Anti-alpha-tubulin</td><td>Sigma-Aldrich</td><td>T-9026</td><td></td></tr><tr><td>Anti-Ezrin</td><td>Sigma-Aldrich</td><td>clone3C12, SAB4200806</td><td></td></tr><tr><td>Anti-Galphaq</td><td>Santacruz</td><td>sc-393</td><td></td></tr><tr><td>Anti-MAP2</td><td>Chemicon</td><td>clone AP20, MAB3418</td><td></td></tr><tr><td>Anti-Moesin</td><td>Sigma-Aldrich</td><td>clone 38/87, M7060</td><td></td></tr><tr><td>Anti-PLCbeta1</td><td>Santacuz</td><td>sc-5291</td><td></td></tr><tr><td>Anti-PSD95</td><td>MA2</td><td>ABR</td><td></td></tr><tr><td>Anti-Spectrin beta</td><td>Chemicon</td><td>MAB1622</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>0080085SA</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo</td><td>23227</td><td></td></tr><tr><td>Bromophenol blue</td><td>Merck</td><td>1.08122.0005</td><td></td></tr><tr><td>Calcium chrolide, hydrous</td><td>Wako</td><td>038-19735</td><td></td></tr><tr><td>Cell scraper</td><td>Falcon</td><td>353085</td><td></td></tr><tr><td>Cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Choline chloride</td><td>Sigma-Aldrich</td><td>C7527</td><td></td></tr><tr><td>Complete EDTA free protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Cytosine beta-D-arabinofuranoside</td><td>Sigma-Aldrich</td><td>C-6645</td><td></td></tr><tr><td>DNase-I</td><td>Sigma-Aldrich</td><td>DN-25</td><td></td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma-Aldrich</td><td>P5155</td><td></td></tr><tr><td>DynaMag-2 Magnet</td><td>Thermo</td><td>12321D</td><td></td></tr><tr><td>ECL Prime Western Blotting Detection Reagent</td><td>GE</td><td>RPN2232</td><td></td></tr><tr><td>e-PAGEL 5-20% SDS-PAGE gradient gel</td><td>ATTO</td><td>E-T520L</td><td></td></tr><tr><td>Folic acid</td><td>Sigma-Aldrich</td><td>F8758</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175095</td><td></td></tr><tr><td>HRP-conjugated anti-rabbit IgG</td><td>Jackson ImmunoResearch</td><td>111-035-144</td><td></td></tr><tr><td>i-Inositol</td><td>Sigma-Aldrich</td><td>I7508</td><td></td></tr><tr><td>LAS-1000 mini</td><td>Fuji Film</td><td>LAS-1000 mini</td><td>For detection of luminescence from WB membrane</td></tr><tr><td>Magnetic polystyrene microbeads</td><td>Sperotech</td><td>PM-20-10</td><td></td></tr><tr><td>MEM amino acid solution</td><td>Gibco</td><td>11130-051</td><td>30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H&lt;sub&gt;2&lt;/sub&gt;O, 20 mM L-Isoleucine, 20 mM L-Leucine, 19.8 mM L-Lysine hydrochloride, 5.1 mM L-Methionine, 10 mM L-Phenylalanine, 20 mM L-Threonine, 2.5 mM L-Tryptophan, 10 mM L-Tyrosine, and 20 mM L-Valine</td></tr><tr><td>Mini-slab size electrophoresis system</td><td>ATTO</td><td>AE-6530</td><td></td></tr><tr><td>Niacinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Penicilin / Streptomycin</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>PhosSTOP phosphatase inhibitor cocktail</td><td>Roche</td><td>4906845001</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Nacali</td><td>28360-14</td><td></td></tr><tr><td>Pyridoxal HCl</td><td>Sigma-Aldrich</td><td>P6155</td><td></td></tr><tr><td>Riboflavin</td><td>Sigma-Aldrich</td><td>R9504</td><td></td></tr><tr><td>Silver Stain 2 Kit wako</td><td>Wako</td><td>291-5031</td><td></td></tr><tr><td>Thiamine HCl</td><td>Sigma-Aldrich</td><td>T1270</td><td></td></tr><tr><td>Trans-Blot SD Semi-Dry Transfer Cell</td><td>Bio-rad</td><td>1703940JA</td><td></td></tr><tr><td>Ultra pure water</td><td>MilliQ</td><td></td><td>For production of ultra pure water</td></tr></tbody></table>

purification of the dendritic filopodia-rich fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In cultured hippocampal neurons, TLCN was abundantly localized to the dendritic filopodia, shaft, and soma and colocalized with F-actin (Figure 1A, B). When polystyrene microbeads were added to cultured hippocampal neurons, the beads were automatically coated with vitronectin (VN) derived from fetal bovine serum (FBS) in the culture medium; they were mainly bound to dendrites, and they induced the formation of phagocytic cups (<strong class="...

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Purification of the Dendritic Filopodia-rich Fraction

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

purification-of-the-dendritic-filopodia-rich-fraction

Research

JoVE Journal

Neuroscience

5.2K Views.  RIKEN Brain Science Institute. In vergelijking met psd-fractie kan het mogelijk zijn om het synaptische eiwit te identificeren dat werkt op de onrijpe synaps rond de dendritische filopodia-rijke fractie. We gebruikten levend neuron om fagocytische cap vorming te induceren. Het belangrijkste voordeel van deze techniek is dat actief eiwit in kunstmatige ontstaan kan worden geïdentificeerd uit de dendritische filopodia rijke fractie.Om te beginnen, bereid 200x vitamine mix door het oplossen van 100 mg vitamine B5, 100...

Video: Purification of the Dendritic Filopodia-rich Fraction

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Biochemistry

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Biology

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Immunology and Infection

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Purification of the Dendritic Filopodia-rich Fraction

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