Neuronal microtubule organization: from minus end to plus end

Neurons are highly polarized cells consisting of a dendritic part and axonal part. Dendrites receive signals from other cells while axons transmit signals to other cells. In this thesis, mostly hippocampal neurons from rat embryos are used to study fundamental aspects of the microtubule organization in neurons, with the aid of molecular biology and microscopy techniques.
Previously it was shown that microtubules can regulate the spine morphology in dendrites and regulate synaptic plasticity. However, little was known about neuronal receptor activation and the effect on the underlying microtubule cytoskeleton. We show that neuronal activity affects the microtubule dynamics in neurons and redistributed EB3 to MAP2 positive microtubules. In another study we found that CAMSAP2 was also mislocalized from its initial position on microtubules after glutamate receptor activation. These results indicate a role for neuronal activity on microtubule binding proteins that influences the dynamics of microtubules.
As neurons develop into highly polarized cells, the neuronal microtubule organization also develops into a highly polarized structure. To create and maintain this polarized structure, a neuron needs to be able to stabilize its microtubules as it does not have a microtubule organizing centre. We found that CAMSAP2 plays a role in stabilizing the free minus ends of microtubules in neurons. Characterization on the function of the family of CAMSAP proteins lead to the conclusion that CAMSAP2 is required for organization and stabilization of interphase microtubules and directional cell migration. Furthermore, CAMSAPs have been found to regulate microtubule minus-end growth and deposite specifically on the growing microtubule minus-end. The length of the stretches that are formed is then regulated by the microtubule severing protein, katanin. In addition, we have shown that the stabilization of microtubules by CAMSAP2 in neurons is needed in order to control neuronal polarity and dendrite development.
In the past, studies using electron microscopy have shed light on the neuronal microtubule organization during neuronal development. Those studies are however based on static images and fixation assays, that may influence the overall mixed polarity of microtubules in dendrites. We used live-cell imaging to visualize dynamic microtubule plus ends. Together with a laser microsurgery technique we were able to probe the neuronal microtubule organization during different developmental stages in hippocampal cultures and brain slices. In vivo imaging also confirmed the bidirectional microtubule organization in dendrites. We have shown that during the different developmental stages of hippocampal neurons in culture, a mixed microtubule organization is observable.