Microtubule retrograde flow retains neuronal polarization in a fluctuating state

In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array itself has been regarded as stationary. Using live cell imaging in combination with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This microtubule retrograde flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation. Shortly after axon formation, microtubule retrograde flow slows down in the axon, which stabilizes microtubule density cycles and thereby functions as a molecular wedge to enable axon extension. We propose microtubule retrograde flow and its specific slowdown in the axon to be the long-sought mechanism to single one neurite out to drive neuronal polarization.

Matrix elasticity gradients guide neuronal polarity by controlling microtubule network mobility

Neuronal polarization and axon specification depend on extracellular cues, intracellular signaling, cytoskeletal rearrangements and polarized transport, but the interplay between these processes has remained unresolved. The polarized transport of kinesin-1 into a specific neurite is an early marker for axon identity, but the mechanisms that govern neurite selection and polarized transport are unknown. We show that extracellular elasticity gradients control polarized transport and axon specification, mediated by Rho-GTPases whose local activation is necessary and sufficient for polarized transport. Selective Kinesin-1 accumulation furthermore depends on differences in microtubule network mobility between neurites and local control over this mobility is necessary and sufficient for proper polarization, as shown using optogenetic anchoring of microtubules. Together, these results explain how mechanical cues can instruct polarized transport and axon specification.

α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation

Tri-methylation on lysine 40 of α-tubulin (α-TubK40me3) is a recently identified post-translational modification involved in mitosis and cytokinesis. However, knowledge about α-TubK40me3 in microtubule function and post-mitotic cells remains largely incomplete. Here, we report that α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation. α-TubK40me3 is enriched in mouse cerebral cortex during embryonic day (E)14 to E16. Knockdown of α-tubulin methyltransferase SETD2 at E14 leads to the defects in neuronal migration, which could be restored by overexpressing either a cytoplasm-localized SETD2 truncation or α-TubK40me3-mimicking mutant. Furthermore, α-TubK40me3 is preferably distributed on polymerized microtubules and potently promotes tubulin nucleation. Downregulation of α-TubK40me3 results in reduced microtubule abundance in neurites and disrupts neuronal polarization, which could be rescued by Taxol. Additionally, α-TubK40me3 is increased after losing α-tubulin K40 acetylation (α-TubK40ac) and largely rescues α-TubK40ac function. This study reveals a critical role of α-TubK40me3 in microtubule formation and neuronal development.

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior–posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

Involvement of JNK1 in Neuronal Polarization During Brain Development

The c-Jun N-terminal Kinases (JNKs) are a group of regulatory elements responsible for the control of a wide array of functions within the cell. In the central nervous system (CNS), JNKs are involved in neuronal polarization, starting from the cell division of neural stem cells and ending with their final positioning when migrating and maturing. This review will focus mostly on isoform JNK1, the foremost contributor of total JNK activity in the CNS. Throughout the text, research from multiple groups will be summarized and discussed in order to describe the involvement of the JNKs in the different steps of neuronal polarization. The data presented support the idea that isoform JNK1 is highly relevant to the regulation of many of the processes that occur in neuronal development in the CNS.

A paralog-specific role of COPI vesicles in the neuronal differentiation of mouse pluripotent cells

Coat protein complex I (COPI)–coated vesicles mediate membrane trafficking between Golgi cisternae as well as retrieval of proteins from the Golgi to the endoplasmic reticulum. There are several flavors of the COPI coat defined by paralogous subunits of the protein complex coatomer. However, whether paralogous COPI proteins have specific functions is currently unknown. Here, we show that the paralogous coatomer subunits γ1-COP and γ2-COP are differentially expressed during the neuronal differentiation of mouse pluripotent cells. Moreover, through a combination of genome editing experiments, we demonstrate that whereas γ-COP paralogs are largely functionally redundant, γ1-COP specifically promotes neurite outgrowth. Our work stresses a role of the COPI pathway in neuronal polarization and provides evidence for distinct functions for coatomer paralogous subunits in this process.