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Purification of the Dendritic Filopodia-rich Fraction.
J Vis Exp 2019; (147)JV

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.

Authors+Show Affiliations

Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute; Liver Cancer Prevention Research Unit, RIKEN Center for Integrative Medical Sciences; yfurutani@riken.jp.Laboratory for Systems Molecular Ethology, RIKEN Center for Brain Science.

Pub Type(s)

Journal Article
Video-Audio Media

Language

eng

PubMed ID

31107457

Citation

Furutani, Yutaka, and Yoshihiro Yoshihara. "Purification of the Dendritic Filopodia-rich Fraction." Journal of Visualized Experiments : JoVE, 2019.
Furutani Y, Yoshihara Y. Purification of the Dendritic Filopodia-rich Fraction. J Vis Exp. 2019.
Furutani, Y., & Yoshihara, Y. (2019). Purification of the Dendritic Filopodia-rich Fraction. Journal of Visualized Experiments : JoVE, (147), doi:10.3791/59292.
Furutani Y, Yoshihara Y. Purification of the Dendritic Filopodia-rich Fraction. J Vis Exp. 2019 May 2;(147) PubMed PMID: 31107457.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification of the Dendritic Filopodia-rich Fraction. AU - Furutani,Yutaka, AU - Yoshihara,Yoshihiro, Y1 - 2019/05/02/ PY - 2019/5/21/entrez JF - Journal of visualized experiments : JoVE JO - J Vis Exp IS - 147 N2 - 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. SN - 1940-087X UR - https://www.unboundmedicine.com/medline/citation/31107457/Purification_of_the_Dendritic_Filopodia-rich_Fraction L2 - https://dx.doi.org/10.3791/59292 DB - PRIME DP - Unbound Medicine ER -