Purification of the Dendritic Filopodia-rich Fraction

Summary

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.

Abstract

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.

Introduction

Dendritic filopodia are thought to be precursors of spines. Actin filaments in the dendritic filopodia regulate their extension and retraction^1,2,3. After contacting with an axon, selected dendritic filopodia begin their maturation into spines, and a synapse is formed^4,5. Components of spines have been determined from comprehensive analysis of postsynaptic density fractions^6,7, 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 formation^5,8,9,10,11, 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 α-actinin in its cytoplasmic region^5,8,14,15,16. TLCN binds to the actin cytoskeleton through phospho-ERM at the tips of dendritic filopodia and α-actinin in spines and dendritic shafts^8,16.

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.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee of RIKEN Wako.

1. Culture of Hippocampal Neurons

1. Preparation of culture medium
   1. 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 °C.
   2. 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 °C.
   3. Preparation of 1 M CaCl₂. Dissolve 7.35 g of CaCl₂·2H₂O in 50 mL of ultrapure water using a magnetic stirrer.
   4. Preparation of Minimum essential medium (MEM). Dissolve 400 mg of KCl, 6800 mg of NaCl, 2,200 mg of NaHCO₃, 158 mg of NaH₂PO₄·2H₂O, 7000 mg of D-glucose, and 200 mg of MgSO₄-7H₂O in 950 mL of ultrapure water using a magnetic stirrer.
   5. 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.
   6. Add 5 mL of 200x Vitamin mix and 200 μL 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 μm filter system and store it at 4 °C.
   7. Preparation of 10x DNase-I stock solutions. Dissolve 100 mg of DNase-I in 12.5 mL of Hanks' Balanced Salt Solution (HBSS), filter through a 0.22 μm filter, aliquot in 1.5 mL tubes, and store the tubes at -20 °C.
   8. Preparation of cytosine β-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 μm filter, aliquot in 1.5 mL tubes and store at -20 °C
   9. Preparation of Plating medium. Mix 1 mL of MEM amino acid solution, 750 μL of 1 M HEPES, 1 mL of B27, 125 μL of 200 mM glutamine, 250 μL of penicillin/streptomycin, 2.5 mL of fetal bovine serum (FBS), and 44.375 mL of MEM in a 50 mL tube.
   10. Preparation of Stop medium. Mix 1 mL of MEM amino acid solution, 750 μL of 1 M HEPES, 5 mL of FBS (final 10%), and 43.25 mL of MEM in a 50 mL tube.
2. Preparation of poly-L-Lysine-coated dishes
   1. Coat 35 mm plastic cell culture dishes with 0.2 mg/mL of poly-L-lysine hydrobromide for 1 day at 25 °C.
      NOTE: Poly-L-lysine should not be used instead of poly-L-lysine hydrobromide.
   2. Wash the dishes with 2 mL of ultrapure water 3 times. Incubate the dishes with 1.5 mL of Stop medium at 25 °C until use.
3. Dissection of hippocampal neurons from mouse embryo
   1. 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.
   2. Incubate dissected hippocampi in 0.25% trypsin and 1x DNaseI in HBSS containing 15 mM HEPES, pH 7.2 for 15 min at 37 °C with agitation every 3 min. Remove the solution. Incubate the hippocampi in 10 mL of STOP medium to inactivate trypsin at 4 °C for 5 min.
   3. Move the hippocampi into 10 mL of fresh STOP medium and incubate at 4 °C for 5 min. Move the hippocampi into 10 mL of fresh STOP medium and incubate at 4 °C for another 5 min. Move the hippocampi into 900 μL of STOP medium and 100 μL 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.
   4. Add 9 mL of plating medium, and filter through a 70 μm 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.
   5. 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).
   6. After 60-64 h of incubation under 5% CO₂ at 37 °C, add 2 μL of Ara-C stock solution (final 10 μM) 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 °C.

2. Purification of Dendritic Filopodia-rich Fraction

1. 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 μL/dish of lysis buffer (PBS containing 0.01% Triton X-100, EDTA-free protease inhibitor cocktail, and phosphatase inhibitor cocktail).
2. 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.
3. Transfer the beads to a new low-protein-binding microtube, set on a magnetic separator, and completely remove the supernatant. Add 500 μL 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 μL of lysis buffer. Repeat the washing of the beads 10 times and remove the supernatant.
4. Elute proteins bound to the beads (the bound fraction) by the addition of 50 μL 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 °C for 5 min. Centrifuge the tube at 860 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.
5. 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/μL for SDS-PAGE.

3. Silver Staining and Western Blot Analysis

1. Separate the bound and unbound fractions (50 ng) by SDS-PAGE using a 5-20% gradient gel. Silver-stain the gel.
2. Western blot using anti-TLCN-C (1/3,000), anti-bovine vitronectin (1/5,000), anti-actin (1/1,000), and anti-α-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.

Results

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 (

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
1 M HEPES	Gibco	15630-080
1.7 ml Low Binding MCT	Sorenson BioScience	39640T
200 mM L-Glutamine	Gibco	2530149
35-mm plastic cell culture dishes	Corning	430165
Anti-actin	Sigma-Aldrich	A-5060
Anti-alpha-Actinin	Sigma-Aldrich	A-5044
Anti-alpha-tubulin	Sigma-Aldrich	T-9026
Anti-Ezrin	Sigma-Aldrich	clone3C12, SAB4200806
Anti-Galphaq	Santacruz	sc-393
Anti-MAP2	Chemicon	clone AP20, MAB3418
Anti-Moesin	Sigma-Aldrich	clone 38/87, M7060
Anti-PLCbeta1	Santacuz	sc-5291
Anti-PSD95	MA2	ABR
Anti-Spectrin beta	Chemicon	MAB1622
B27	Gibco	0080085SA
BCA protein assay kit	Thermo	23227
Bromophenol blue	Merck	1.08122.0005
calcium chrolide, hydrous	Wako	038-19735
Cell scraper	Falcon	353085
Cell strainer	Falcon	352350
Choline chloride	Sigma-Aldrich	C7527
Complete EDTA free protease inhibitor cocktail	Roche	11873580001
Cytosine beta-D-arabinofuranoside	Sigma-Aldrich	C-6645
DNase-I	Sigma-Aldrich	DN-25
D-Pantothenic acid hemicalcium salt	Sigma-Aldrich	P5155
DynaMag-2 Magnet	Thermo	12321D
ECL Prime Western Blotting Detection Reagent	GE	RPN2232
e-PAGEL 5-20% SDS-PAGE gradient gel	ATTO	E-T520L
Folic acid	Sigma-Aldrich	F8758
HBSS	Gibco	14175095
HRP-conjugated anti-rabbit IgG	Jackson ImmunoResearch	111-035-144
i-Inositol	Sigma-Aldrich	I7508
LAS-1000 mini	Fuji Film	LAS-1000 mini	For detection of luminescence from WB membrane
Magnetic polystyrene microbeads	Sperotech	PM-20-10
MEM amino acid solution	Gibco	11130-051	30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H2O, 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
Mini-slab size electrophoresis system	ATTO	AE-6530
Niacinamide	Sigma-Aldrich	N0636
Penicilin / Streptomycin	Gibco	15070063
PhosSTOP phosphatase inhibitor cocktail	Roche	4906845001
Poly-L-lysine hydrobromide	Nacali	28360-14
Pyridoxal HCl	Sigma-Aldrich	P6155
Riboflavin	Sigma-Aldrich	R9504
Silver Stain 2 Kit wako	Wako	291-5031
Thiamine HCl	Sigma-Aldrich	T1270
Trans-Blot SD Semi-Dry Transfer Cell	Bio-rad	1703940JA
Ultra pure water	MilliQ		For production of ultra pure water