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 t....

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 Riboflav....

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