Roles of motor proteins in cerebellar Purkinje neuron biology

National Heart, Lung, And Blood Institute

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Abstract

Myosin 18A is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18A is -Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A. Importantly, -Pix colocalizes with myosin 18A in spines, and its spine localization is lost upon myosin 18A knockdown or when its myosin 18A binding site is deleted. Finally, we show that the spines of myosin 18A knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A form a complex with -Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of -Pix's GEF activity. Myosin 10 (Myo10) is a highly-conserved, vertebrate-specific unconventional myosin whose tail domain contains a PIP3-specific PH domain, a microtubule-binding MyTH4 domain, and an integrin-binding FERM domain (Kerber and Cheney, 2011). Myo10 has been linked primarily to the formation and maintenance of filopodia (it is commonly referred to as the filopodial myosin), the transport of integrins to the tips of filopodia, pathfinding during cell migration, and the positioning of mitotic and meiotic spindles. Interestingly, Myo10 has also been shown to function in radial neuron migration (Ju et al, 2014) and in the transport of the netrin-1 receptor DCC to the tips of neurites to regulate axonal pathfinding (Zhu et al, 2007). Our past efforts to define the functions of another unconventional myosin (myosin Va) in cerebellar Purkinje neurons (PN), the master neuron of the cerebellum, led to the development of novel tools to study this complex neuron (Wagner et al, 2011; Alexander and Hammer, 2016, 2019). Here we describe ongoing efforts using these tools and a Myo10 knockout (KO) mouse we created (Heimsath et al, 2017) to define the function of Myo10 in PNs and in the cerebellum. Our decision to pursue Myo10 function in PNs was based on two linked observations. First, PNs are unique among CNS in possessing very high levels of Myo10 mRNA. Consistently, Western blots show that the amount of Myo10 in the cerebellum increases dramatically during the organs postnatal development, and remains high in adulthood. Second, PNs are also unique among CNS neurons in that their dendritic spines develop normally from filopodial precursors in the absence of innervation. One possibility, therefore, is that filopodia-to-spine conversion can occur in PNs without innervation because they express high levels of this filopodial myosin. Here we describe our observations to date regarding the function of Myo10 in PNs and the cerebellum. First, 6 week-old Myo10 KO mice (i.e. young adults) exhibit cerebellar hypoplasia and the abnormal formation of cerebellar lobes (misshapen and/or missing). Second, Calbindin staining of cerebellar slices from these mice shows that PNs exhibit defects in the orientation of their dendritic arbor within the molecule layer and in the alignment of their soma. Third, Calbindin staining of cerebellar slices from 6 month-old Myo10 KO mice (i.e. mature adults) shows that PNs exhibit significant reductions in dendritic arborization and spine density (these results are currently being confirmed using Golgi-Cox staining). Fourth, 6 week-old Myo10 KO mice exhibit significant defects in balance and other behavioral tests of cerebellar function (behavioral analyses of 6 month-old Myo10 KO mice are underway). Fifth, preliminary experiments show that the miRNA-mediated KD of Myo10 in cultured PNs results in defects in spine maturation and cell polarity (increased number of axons, reduced dendritic arborization). Finally, GFP- tagged Myo10 expressed in cultured PNs localizes dramatically to the tips of filopodia at the leading edge of developing neurites (this imaging is now being extended using PNs isolated from a GFP-Myo10 knockin mouse we recently created). Together, these results indicate that Myo10 is required for normal cerebellar development, cerebellar function, and PN structure and function, and they pave the way for future efforts designed to identify the molecular mechanisms by which Myo10 promotes these processes.