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Building the Neuronal Microtubule Cytoskeleton

Final Report Summary - NEUROMT (Building the Neuronal Microtubule Cytoskeleton)

The human brain consists of more than one hundred billion neurons, intricately connected into functional neuronal circuits. The formation of complex nervous systems requires processes that coordinate proliferation, migration and differentiation of neuronal cells. The remarkable morphological transformations of neurons as they migrate, extend axons and dendrites and establish synaptic connections, imply a strictly regulated process of structural organization and dynamic remodeling of the cytoskeleton. Pioneering work using embryonic hippocampal cultures established a paradigm in which isolated neurons defined the morphological steps of polarization, differentiation and spine formation. Careful analysis of these in vitro models led to the observation that neurons transition through several developmental stages, from freshly plated stage 1 cells bearing immature neurites to stage 5 cells that exhibit mature axons, dendrites, dendritic spines, and functional synapses. The actin and MT cytoskeleton have a pivotal role at all stages; together they determine neuronal architecture, organize cellular compartments and transport intracellular constituents. In this ERC program, we have addressed three important questions about how the MT cytoskeleton is formed and organized at different phases of neuronal development. What is the role of the centrosome in MT nucleation in the early stages of neuronal development? What is the mechanism by which dendrites organize MTs into bipolar arrays? What is the relation between MT spine entry and postsynaptic cargo transport?

I will give three research highlights that came out of this ERC research program:

1/ We show that neuronal migration, development, and polarization depend on the multi-subunit protein HAUS/augmin complex, previously described to be required for mitotic spindle assembly in dividing cells. The HAUS complex is essential for neuronal microtubule organization by ensuring uniform microtubule polarity in axons and regulation of microtubule density in dendrites. Using live-cell imaging and high-resolution microscopy, we found that distinct HAUS clusters are distributed throughout neurons and colocalize with γ-TuRC, suggesting local microtubule nucleation events. We propose that the HAUS complex locally regulates microtubule nucleation events to control proper neuronal development.

2/ By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubule-binding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.

3/ To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2. We showed that calcium, acting via CaM, enhances KIF1A binding to DCVs and increases vesicle motility. In contrast, liprin-α and TANC2 are not part of the KIF1A-cargo complex but capture DCVs at dendritic spines. We propose a model in which Ca2+/CaM regulates cargo binding and liprin-α and TANC2 recruit KIF1A-transported vesicles

In this ERC research program, new discoveries have advanced our understanding of how basic cellular mechanisms are responsible for organizing the microtubule cytoskeleton during neuronal development. The integrated, quantitative understanding of the interplay between cytoskeleton organization and cargo transport in neurons has given us new insights in neuronal polarity, neuronal development and synaptic processes.