Furutani Yutaka, Yoshihara Yoshihiro
Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute; Liver Cancer Prevention Research Unit, RIKEN Center for Integrative Medical Sciences;
Laboratory for Systems Molecular Ethology, RIKEN Center for Brain Science.
J Vis Exp. 2019 May 2(147). doi: 10.3791/59292.
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.
树突丝状伪足是基于肌动蛋白丝的细长突起，它们会伸展和收缩，仿佛在寻找目标轴突。当树突丝状伪足与目标轴突建立接触时，它们开始成熟为棘突，从而导致突触的形成。端脑蛋白（TLCN）大量定位于树突丝状伪足中，并逐渐从棘突中排除。在培养的海马神经元中过表达TLCN会诱导树突丝状伪足的形成。我们发现端脑蛋白与细胞外基质分子玻连蛋白强烈结合。包被有玻连蛋白的微珠会在神经元树突上诱导吞噬杯的形成。在吞噬杯中，TLCN、诸如磷酸化埃兹蛋白/根蛋白/膜突蛋白（磷酸化ERM）等TLCN结合蛋白以及F-肌动蛋白会聚集，这表明吞噬杯的成分与树突丝状伪足的成分相似。因此，我们开发了一种纯化吞噬杯而非树突丝状伪足的方法。用玻连蛋白包被磁性聚苯乙烯珠，玻连蛋白大量存在于海马神经元的培养基中，且会在神经元树突上诱导吞噬杯的形成。孵育24小时后，用去污剂温和地溶解吞噬杯，并用磁力分离器收集。洗涤珠子后，洗脱结合蛋白，并通过银染和蛋白质印迹法进行分析。在结合组分中，大量存在TLCN和肌动蛋白。此外，从该组分中鉴定出的许多蛋白质定位于树突丝状伪足；因此，我们将结合组分命名为富含树突丝状伪足的组分。本文描述了关于富含树突丝状伪足组分纯化方法的详细信息。
