MST3 Kinase Phosphorylates TAO1/2 to Enable Myosin Va Function in Promoting Spine Synapse Development

Sila K Ultanir,Smita Yadav,Nicholas T Hertz,Juan A Oses-Prieto,Suzanne Claxton,Alma L Burlingame,Kevan M Shokat,Lily Y Jan,Yuh-Nung Jan

doi:10.1016/j.neuron.2014.10.025

Sila K Ultanir, Smita Yadav + Show 7 more

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https://doi.org/10.1016/j.neuron.2014.10.025

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SummaryMammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.

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Dendrite arborization and synapse formation are critical for wiring the neural circuitry (Jan and Jan, 2010; Parrish et al, 2006)
MST3 Limits Filopodia on Developing Dendrites Localization of endogenous MST3 in punctate structures in the neuronal soma and dendrites was evident from immunofluorescence staining of dissociated hippocampal neurons double labeled with the dendrite marker microtubule-associated protein 2 (MAP2) (Figure 1A), as well as immunofluorescence staining of GFP transfected neurons in high density cultures with monoclonal antibody against MST3 (Figure 1B)
Inhibiting MST3 function by expressing either MST3 shRNA or MST3 kinase dead (K53R) mutant (MST3-KD) together with GFP resulted in a dramatic increase in the total dendritic filopodia length per 50 mm dendrite and filopodia density (Figures 1C and 1D), as well as average filopodia length (Figure S1C)

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Dendrite arborization and synapse formation are critical for wiring the neural circuitry (Jan and Jan, 2010; Parrish et al, 2006). The predominant excitatory neurons in the mammalian brain, contain dendritic spines, postsynaptic structures harboring more than 90% of excitatory synapses in the brain (Harris and Kater, 1994; Nimchinsky et al, 2002). Dendritic spine formation is preceded by actinrich filopodia that typically contain immature synapses and are thought to be involved in dendrite arborization and synaptogenesis (Fiala et al, 1998; Yuste and Bonhoeffer, 2004). Unraveling the molecular mechanisms underlying spine formation is an important research area. A better understanding of the molecular mechanisms involved in brain development and synapse formation could enable future therapeutic interventions

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