Distinct Roles for Dynein Regulatory Proteins NudE and NudEL in Brain Development

The development of the mammalian neocortex requires the careful balancing of proliferation, migration, and differentiation. The cellular machinery coordinating these events includes molecular motor proteins such as dynein. Regulation of dynein activity is particularly important, since it is the major microtubule minus-end directed motor in cells. Dynein is a large, complex structure comprising several subunits and binding partners. Its function is critical for multiple stages of brain development. The dynein regulatory proteins NudE and NudEL have been implicated in several aspects of dynein function, including brain development. Originally identified as nuclear distribution (nud) factors in the dynein pathway, NudE and NudEL are now known to have diverse roles in mitosis, cell migration, and intracellular trafficking. Mice null for Nde1, the gene encoding NudE, have microcephaly, whereas mice null for Ndel1, which encodes NudEL, are embryonic lethal. Additionally, Nde1 mutations have recently been shown to result in microcephaly and lissencephaly in human patients. NudE and NudEL are functionally related paralogs that are more than 70% similar. Both bind to dynein and LIS1, another dynein regulatory protein involved in brain development. In addition to serving as recruitment factors, NudE and NudEL impact dynein force production and allow dynein to serve as a persistent motor under high load. This would be particularly important during the proliferation of neural progenitors, which undergo cell cycle-linked nuclear oscillations. These oscillations, termed interkinetic nuclear migration (INM), require forces acting upon the nucleus to drive upward (basal) and downward (apical) movement in the proliferative ventricular zone (VZ) of the brain. Research from our lab has identified dynein, along with LIS1, as being responsible for apical movement, and the unconventional kinesin Kif1a as the driving force behind basal movement. The aim of this thesis has been to understand the mechanisms by which NudE and NudEL regulate dynein function in brain development. We identify a role for NudE, but not NudEL, in INM and radial progenitor mitosis. Additionally, we find that both NudE and NudEL are involved in the multipolar-to-bipolar transition of neurons, and that NudEL has a role in bipolar neuronal migration. Our results provide an additional molecular explanation for microcephaly resulting from Nde1 mutations, implicating a block in INM as a cause for reduced proliferation, since cells are unable to reach the ventricular surface where they normally undergo mitosis. NudEL has previously been implicated in having a role in neurite extension and axon elongation. We found that NudE/EL localized to a single neurite of a Stage 2 hippocampal neuron as well as the axon tip of a Stage 3 neuron. In addition, the Stage 2 localization was coincident with the appearance of established early markers of neuronal polarity. We studied the role of NudE/EL in establishing neuronal polarity and found that Nde1 and Ndel1 RNAi inhibited axon formation. Overexpression of NudEL did not result in noticeable changes in axon formation. We conclude that in addition to the role of NudEL in axon extension and outgrowth, NudE/EL serve as early markers of neuronal polarity and are required, though not necessarily sufficient, for axon specification

Severe NDE1-mediated microcephaly results from neural progenitor cell cycle arrests at multiple specific stages

Microcephaly is a cortical malformation disorder characterized by an abnormally small brain. Recent studies have revealed severe cases of microcephaly resulting from human mutations in the NDE1 gene, which is involved in the regulation of cytoplasmic dynein. Here using in utero electroporation of NDE1 short hairpin RNA (shRNA) in embryonic rat brains, we observe cell cycle arrest of proliferating neural progenitors at three distinct stages: during apical interkinetic nuclear migration, at the G2-to-M transition and in regulation of primary cilia at the G1-to-S transition. RNAi against the NDE1 paralogue NDEL1 has no such effects. However, NDEL1 overexpression can functionally compensate for NDE1, except at the G2-to-M transition, revealing a unique NDE1 role. In contrast, NDE1 and NDEL1 RNAi have comparable effects on postmitotic neuronal migration. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage

Dynamics of Dynamin during Clathrin Mediated Endocytosis in PC12 Cells

Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both.The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis.This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles

ER and Golgi trafficking in axons, dendrites, and glial processes.

Both neurons and glia in mammalian brains are highly ramified. Neurons form complex neural networks using axons and dendrites. Axons are long with few branches and form pre-synaptic boutons that connect to target neurons and effector tissues. Dendrites are shorter, highly branched, and form post-synaptic boutons. Astrocyte processes contact synapses and blood vessels in order to regulate neuronal activity and blood flow, respectively. Oligodendrocyte processes extend toward axons to make myelin sheaths. Microglia processes dynamically survey their environments. Here, we describe the local secretory system (ER and Golgi) in neuronal and glial processes. We focus on Golgi outpost functions in acentrosomal microtubule nucleation, cargo trafficking, and protein glycosylation. Thus, satellite ER and Golgi are critical for local structure and function in neurons and glia

Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear migration in neural stem cells.

Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration

Different phases of dynamin intensity decrease at sites of clathrin-mediated endocytosis in PC12 cells.

<p>PC12 cells co-transfected with clathrin-dsRed and dynamin1-EGFP (A) or dynamin2-EGFP (B) were imaged by live-cell TIR-FM. Decreases in dynamin fluorescence were aligned so that the peak was set to time 0, and clathrin and Dynamin fluorescence at time 0 were normalized to 1. (B and C) demonstrate the decreases in clathrin and dynamin1 or dynamin2, at the sites of 30 or 28 disappearing clathrin spots, respectively. In each case, the decrease in clathrin intensity fits a single exponential, while both dynamin1 and dynamin2 fit double exponentials (all r² values >0.98).</p