| 研究実績の概要 |
Dendritic filopodia are actin-rich protrusions from the dendrites of neurons that act as both environmental sensors and precursors of future spines. In this study,I investigate the regulation of the actin cytoskeletal network in the dendritic filopodia of Purkinje cells, a neuron with complex dendritic branching, and attempt to delineate the contribution of filopodial morphology to final dendritic morphology. MTSS1 is an actin-binding protein that has high and developmentally-regulated expression in Purkinje cells.I showed that loss of MTSS1 resulted in elongated filopodia, which was rescued by acute inhibition of the actin-nucleating formin family. Overexpression of MTSS1 led to round, bulbous spines which could be rescued with inhibition of the ARP2/3 complex. These opposing changes in dendritic filopodia morphology suggested that in the limited volume of a dendritic filopodia, the two major actin nucleating pathways may compete with free G-actin and MTSS1 may function in this intersection.To better understand how MTSS1 is affecting this balance in actin nucleation,I further identified the direct interaction of MTSS1 with the formin DAAM1 in the mammalian brain and identified the functional domains involved by biochemical experiments.Molecular studies in NIH3T3 cells demonstrated the negative regulation of constitutive active DAAM1-mediated F-actin increase by MTSS1. Together, these results suggest that MTSS1 may partially inhibit DAAM1 in dendritic protrusions during development, and in turn upregulate the ARP2/3 pathway.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We initially aimed to identify MTSS1 binding proteins in neurons. I identified the direct interaction of the actin-binding protein DAAM1 with MTSS1 in the dendritic filopodia and spines of Purkinje cells. Further, I identified which domains of both proteins were necessary for this interaction. Because studies of the regulation of actin nucleators in dendritic filopodia and spines are very limited, we observed the effect of acute inhibition of different actin nucleators on dendritic filopodia morphology and could successfully visualize the competing activities of the two major nucleation pathways in the dendritic filopodia of Purkinje cells.
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| 今後の研究の推進方策 |
In this study we identified the negative regulation of DAAM1 by MTSS1, and identified the domains within each protein responsible for this interaction. We further plan to clarify how MTSS1 inhibits DAAM1 by biochemical in vitro experiments and single-molecule imaging. In our previous observations,because we also observed a dendritic phenotype with loss of MTSS1,we plan to observe the dendritic phenotype resulting from changes in the length of dendritic filopodia.
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