Title: Roles for Cytoplasmic Dynein and the Unconventional Kinesin, KIF1a, during Cortical Development
Description: Radial glial progenitor (RGP) cells are neural stem cells that give rise to the majority of neurons, glia, and adult stem cells during cortical development. These cells divide either symmetrically to form two daughter RGP cells or asymmetrically to form a daughter RGP cell or a daughter neuron/neuronal precursor. In between divisions, the nuclei of RGP cells oscillate in coordination with the cell cycle in a form of behavior known as interkinetic nuclear migration (INM). RGP nuclei migrate basally during G1, undergo S phase, and migrate apically during G2 to the apical, ventricular surface (VS). Mitosis only occurs when the nucleus reaches the VS. Two microtubule-associated motor proteins are required to drive nuclear movement: the unconventional kinesin, Kif1a, during G1-specific basal migration and cytoplasmic dynein during G2-specific apical migration. The strict coordination of motor activity, migratory direction, and cell cycle phase is highly regulated and we find that a G2 cell cycle-dependent protein kinase activates two distinct G2-specific mechanisms to recruit dynein to nuclear pores.

The activities of these pathways initiate apical nuclear migration and maintain nuclear movement throughout G2. Originally identified in HeLa cells, we find the two G2-specific recruitment pathways (“RanBP2-BicD2” and “Nup133-CENP-F”) are conserved in RGP cells. Disrupting either pathway arrests apical nuclear migration but does not affect G1-dependent basal migration. The “RanBP2-BicD2” pathway initiates early during G2 and is maintained throughout the cell cycle phase while the “Nup133-CENP-F” pathway is activated later in G2. Forced targeting of dynein to the nuclear envelope (NE) restores apical nuclear migration, with nuclei successfully reaching the VS. We also find that the G2/M-specific Cdk1 serves as a master regulator of apical nuclear migration in RGP cells. Pharmacological drug inhibitors of Cdk1 arrest apical migration without any effect on G1-dependent basal migration. Conversely, overactivating Cdk1 causes premature, accelerated apical nuclear migration. Specifically, Cdk1 drives apical nuclear migration through activation of both the “RanBP2-BicD2” and “Nup133-CENP-F” pathways.

Cdk1 acts by phosphorylating RanBP2, priming it for BicD2 interaction. Forced targeting of BicD2-dynein to the NE in a RanBP2-independent manner rescues apical nuclear migration in the presence of Cdk1 drug inhibition. Additionally, Cdk1 seems to activate the “Nup133-CENP-F” at the CENP-F level, phosphorylating the protein to trigger nuclear export. INM plays an important role in proper cell cycle progression and we find that arresting nuclei away from the VS prevents mitotic entry, demonstrating that apical nuclear migration to the VS is not just a correlated with cell cycle progression, but is required. When apical migration is restored by forced recruitment of dynein to the NE, mitotic entry is restored as well. In contrast, we find that arresting basal migration by Kif1a does not have a major influence on cell cycle progression. RGP cells still enter S-phase despite remaining close to the VS, revealing that, unlike mitotic entry, S-phase entry is not coupled with nuclear positioning.

However, symmetric, proliferative divisions are favored over asymmetric, neurogenic divisions after inhibition of basal migration. We further find that Kif1a and the proteins involved in the two recruitment pathways play additional role later in brain development. After a neurogenic division, the newly-born neuron migrates past the RPG nuclei and they undergo a multipolar morphology. After at least twenty-four hours, the immature neuron then transitions to a bipolar, migratory morphology where it continues migrating towards its final destination along RGP fibers to the cortical plate. We demonstrate that Kif1a and NE dynein recruitment proteins seem to be involved in the multipolar to bipolar transition and RNAi for these proteins prevent further migration by arresting the immature neurons in a multipolar morphology. Kif1a RNAi, in particular, also induced comparable arrest in surrounding control neurons. Further analysis reveal that the multipolar arrest in neurons is independent of the basal nuclear migration arrest in RGP cells.

These results identify the control mechanism for NE dynein recruitment in RGP cells to drive apical nuclear migration, the relationship of cell cycle phase progression with nuclear positioning, and the sequential, independent roles of these proteins, particularly Kif1a, in neuronal maturation.