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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 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.......

Representative 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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1 M HEPESGibco15630-080
1.7 ml Low Binding MCTSorenson BioScience39640T
200 mM L-GlutamineGibco2530149
35-mm plastic cell culture dishesCorning430165
Anti-actinSigma-AldrichA-5060
Anti-alpha-ActininSigma-AldrichA-5044
Anti-alpha-tubulinSigma-AldrichT-9026
Anti-EzrinSigma-Aldrichclone3C12, SAB4200806
Anti-GalphaqSantacruzsc-393
Anti-MAP2Chemiconclone AP20, MAB3418
Anti-MoesinSigma-Aldrichclone 38/87, M7060
Anti-PLCbeta1Santacuzsc-5291
Anti-PSD95MA2ABR
Anti-Spectrin betaChemiconMAB1622
B27Gibco0080085SA
BCA protein assay kitThermo23227
Bromophenol blueMerck1.08122.0005
calcium chrolide, hydrousWako038-19735
Cell scraperFalcon353085
Cell strainerFalcon352350
Choline chlorideSigma-AldrichC7527
Complete EDTA free protease inhibitor cocktailRoche11873580001
Cytosine beta-D-arabinofuranosideSigma-AldrichC-6645
DNase-ISigma-AldrichDN-25
D-Pantothenic acid hemicalcium saltSigma-AldrichP5155
DynaMag-2 MagnetThermo12321D
ECL Prime Western Blotting Detection ReagentGERPN2232
e-PAGEL 5-20% SDS-PAGE gradient gelATTOE-T520L
Folic acidSigma-AldrichF8758
HBSSGibco14175095
HRP-conjugated anti-rabbit IgGJackson ImmunoResearch111-035-144
i-InositolSigma-AldrichI7508
LAS-1000 miniFuji FilmLAS-1000 miniFor detection of luminescence from WB membrane
Magnetic polystyrene microbeadsSperotechPM-20-10
MEM amino acid solutionGibco11130-05130 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 systemATTOAE-6530
NiacinamideSigma-AldrichN0636
Penicilin / StreptomycinGibco15070063
PhosSTOP phosphatase inhibitor cocktailRoche4906845001
Poly-L-lysine hydrobromideNacali28360-14
Pyridoxal HClSigma-AldrichP6155
RiboflavinSigma-AldrichR9504
Silver Stain 2 Kit wakoWako291-5031
Thiamine HClSigma-AldrichT1270
Trans-Blot SD Semi-Dry Transfer CellBio-rad1703940JA
Ultra pure waterMilliQFor production of ultra pure water

References

  1. Fiala, J. C., Feinberg, M., Popov, V., Harris, K. M. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. Journal of Neuroscience. 18 (21), 8900-8911 (1998).
  2. Portera-Cailliau, C., Pan, D. T., Yuste, R.

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