purification-of-the-dendritic-filopodia-rich-fraction

Purification of the Dendritic Filopodia-rich Fraction

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 ...

RIKEN Brain Science Institute

RIKEN Center for Integrative Medical Sciences

Dendritic filopodia are long, thin, actin-rich, and dynamic protrusions that are thought to play a critical role as a precursor of spines during neural development. We reported previously that a telencephalon-specific cell adhesion molecule, telencephalin (TLCN) [intercellular adhesion molecule-5 (ICAM-5)], is highly expressed in dendritic filopodia, facilitates the filopodia formation, and slows spine maturation. Here we demonstrate that TLCN cytoplasmic region binds ERM (ezrin/radixin/moesin) family proteins that link membrane proteins to actin cytoskeleton. In cultured hippocampal neurons, phosphorylated active forms of ERM proteins are colocalized with TLCN in dendritic filopodia, whereas alpha-actinin, another binding partner of TLCN, is colocalized with TLCN at surface membranes of soma and dendritic shafts. Expression of constitutively active ezrin induces dendritic filopodia formation, whereas small interference RNA-mediated knockdown of ERM proteins decreases filopodia density and accelerates spine maturation. These results indicate the important role of TLCN-ERM interaction in the formation of dendritic filopodia, which leads to subsequent synaptogenesis and establishment of functional neural circuitry in the developing brain.

Interaction between telencephalin and ERM family proteins mediates dendritic filopodia formation.

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.

Dendritic filopodia are thin, long, and highly mobile protrusions functioning as spine precursors. By contrast with a wealth of knowledge on molecular profiles in spines, little is known about structural and functional proteins present in dendritic filopodia. To reveal the molecular constituents of dendritic filopodia, we developed a new method for biochemical preparation of proteins enriched in dendritic filopodia, by taking advantage of specific and strong binding between a dendritic filopodial membrane protein, telencephalin, and its extracellular matrix ligand, vitronectin. When vitronectin-coated magnetic microbeads were added onto cultured hippocampal neurons, phagocytic cup-like membrane protrusions were formed on dendrites through the binding to telencephalin. Magnetically purified membrane protrusion fraction was subjected to comprehensive mass spectrometric analysis and 319 proteins were identified, many of which were confirmed to be localized to dendritic filopodia. Thus, this study provides a useful resource for studying molecular mechanisms underlying dendritic development, synapse formation, and plasticity.

Yutaka Furutani

Laboratory for Neurobiology of Synapse

RIKEN Brain Science Institute

Purification of the Dendritic Filopodia-rich Fraction

Yutaka Furutani

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RIKEN Brain Science Institute

Purification of the Dendritic Filopodia-rich Fraction

Laboratory for Neurobiology of Synapse