Microtubule organisation and polarized transport of kinesins in neurons

The human brain consists of approximately 100 billion neurons that are interconnected to form functional neuronal circuits. The ability of neurons to receive, process and transmit information relies on their polarized organisation into axons and dendrites. This high degree of polarization and the underlying functional compartmentalization present major challenges for sorting and distribution of sub-cellular components. Impairment of intracellular transport has been linked to certain neurodegenerative diseases such as Alzheimer’s disease and Amyotrophic Lateral Sclerosis. Therefore, understanding the molecular mechanisms that govern polarized transport is a central challenge in the field of neurobiology. Microtubules (MTs) are polarized cytoskeletal polymers important for the proliferation, morphology, and intracellular organisation of cells because they support cell division, polarization and intracellular transport. MTs serve as tracks for both kinesin and dynein motor proteins, which move in opposite directions towards the plus- and minus-end of the MT, respectively. Therefore, in neurons, the MT cytoskeleton enables distinct motor proteins to transport cargoes selectively into either axons or dendrites. Whereas axonal MTs are uniformly oriented, dendritic MTs in mammalian neurons have mixed orientations; and remarkably, most MTs in mature neurons are not connected to the centrosome, the main MT organizing centre (MTOC) in other cell types. However, the molecular mechanisms regulating non-centrosomal MTs nucleation and positioning and therefore proper MT polarity in different neuronal compartments have remained unclear. Furthermore, several plus-end directed motor proteins are selective for the axon, while others target both the axon and dendrites. It is widely assumed that these fundamental differences in selectivity are encoded by the MT network; however, the molecular organisation design that ensure axon-selective transport have remained unresolved. By combining molecular engineering, live cell imaging and super-resolution microscopy, we aimed at determining the connection between microtubule organisation and polarized transport in neurons.

In this project we developed an innovative optical nanoscopy technique to examine the relation between MT orientations, stability and modifications in neurons. Using nanometric tracking of motor proteins running over an extracted cytoskeleton to super-resolve MTs and determine their polarity, we find that: i- dendritic MTs are organized in bundles of preferred polarity locally biasing transport; ii- oppositely oriented MT bundles differ in stability and composition; iii- minus-end out MTs are more stable and more acetylated; iv- Kinesin-1 preferentially binds to these minus-end out oriented MTs. Altogether, this explains why the plus-end directed motor Kinesin-1 cannot drive cargo transport into dendrites biasing transport towards the axon. Thus, the spatial segregation of dendritic MTs by orientation, stability and chemical modification uncovered in this project constitutes a key architectural principle of the neuronal MT cytoskeleton that enables selective sorting by different motor proteins. Furthermore, we show that the microtubule nucleation complex HAUS (a microtubule-associated protein) is required for axon development and specification and for maintenance of MT polarity in axons. We show that, in addition to regulation of axonal development, the HAUS complex plays a critical role in dendrite development and regulation of MT density in dendrites while we observe no change in MT polarity. We further characterized HAUS subunits organisation and dynamics in neurons and found that the HAUS complex forms discrete puncta throughout the soma and dendrites, a fraction of which engages in MT nucleation. Finally, by performing mass spectrometry of HAUS subunits, live imaging and super-resolution microscopy we suggest that in neurons maintenance of the HAUS complex requires both MT and F-actin.
Exploitation and dissemination: regarding this project, 6 seminars were given to the Department during my Marie Sklodowska-Curie fellowship. The work performed in the last two years will lead to two publications. First part of the work has already generated wide-spread enthusiasm at the various cell biology, neuroscience and advanced microscopy meetings where it has been presented (e.g. the EMBO conference on Microtubules, the European Neuroscience Meeting (FENS), the GRC on molecular motors, the EMBO Meeting, the European Microscopy Conference); and is under review in Neuron. The second part of this work is now finished and in preparation to be submitted in an open source high impact journal.

Understanding the connection between MT organisation and polarized transport is a major ambition of neurobiology. Altogether, this post-doctoral work revealed how microtubule cytoskeleton organisation underlies neuron development, polarity, morphology and intracellular transport. By using novel super-resolution methodology to directly resolve microtubule orientations and modifications, we have now solved this decades-old problem. We made the surprising discovery that the microtubules preferred by Kinesin-1 are minus-end out oriented in dendrites, whereas those preferred by Kinesin-3 have the opposite orientation. This organisation directly explains for the first time why Kinesin-1 exclusively enters axons, while Kinesin-3 enters both axons and dendrites. In addition, it reveals that Kinesin-1 would operate as a retrograde motor in dendrites. Furthermore, future projects and collaborations will allow this approach to be used in many different model systems to unravel how cytoskeletal organisation underlies organelle positioning, cell morphology and cell division. Furthermore, by using in vivo and in vitro approaches, mass spectrometry, live imaging and super-resolution, we have now deciphered the molecular mechanisms regulating non-centrosomal MTs nucleation in neurons. Here we show for the first time that a microtubule-associated protein, the HAUS complex, is required for axon and dendrite development and cytoskeleton organisation. In addition, we provided the first insights into the subcellular organisation and dynamics of the HAUS complex in neurons and made the surprising discovery that F-actin is involved the regulation of these processes.
This work provides fundamental knowledge required to understand the molecular mechanisms involved in neurodegenerative diseases such as Alzheimer’s disease and Amyotrophic Lateral Sclerosis. Therefore, it will contribute to a better understanding of brain functioning, meeting perfectly the priorities of the current research framework in the European Research Area. The outcome of this study might also provide new strategic approaches to counter such diseases or decrease the consequences for the concerned individuals.

Periodic Reporting for period 1 - KINSELECT (Microtubule organisation and polarized transport of kinesins in neurons)

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