Abstract
Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron’s ‘two-stroke’ nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation.
Highlights
Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits
In cerebellar granule neurons (CGNs), both the actin and microtubule cytoskeletal motor systems contribute to two-stroke motility (Supplementary Note 1, Supplementary Figs 1 and 2); we explored functional microtubule–actomyosin interactions as a means to define mechanistically how both systems work together to impact neuronal motility
We reasoned that drebrin would be a useful reporter for establishing the sites of microtubule–actomyosin interaction in migrating CGNs, as it binds the sides of f-actin filaments and microtubule plus ends through direct interaction with the neuronal þ TIP protein end-binding protein 3 (EB3)[41,47]
Summary
Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. We examined the cell biological underpinnings of microtubule–actomyosin crosstalk in cerebellar granule neurons (CGNs) by utilizing drebrin, a protein that links microtubule movements to the actomyosin cytoskeleton in the growth cone and synapse[41,42,43], as a molecular entry point[44]. We applied advanced lattice light-sheet (LLS) microscopy and Super-resolution structured illumination microscopy (SR-SIM) in combination with functional studies examining the dynamic localization and function of drebrin in migrating CGNs. We applied advanced lattice light-sheet (LLS) microscopy and Super-resolution structured illumination microscopy (SR-SIM) in combination with functional studies examining the dynamic localization and function of drebrin in migrating CGNs These studies reveal that drebrin and f-actin in the proximal leading process/cytoplasmic dilation constitute a novel interaction interface directing movements of the centrosome and microtubules that steer the polarity of the two-stroke motility cycle. We define a novel functional antagonism between drebrin and the Siah[2] E3 ubiquitin ligase that illustrates how the cytoskeletal underpinnings of the two-stroke motility cycle are regulated during neuronal differentiation
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