Cellular Logistics: Form, Formation and Function of the Neuronal Microtubule Cytoskeleton

The organization and dynamics of the MT (MT) cytoskeleton underlies the morphology, polarization and division of most cells. The structural polarity of MT determines the directionality of motor proteins, which move selectively towards either the MT plus (most kinesins) or minus end (dynein) to control the transport and positioning of proteins and organelles. Understanding how different cellular MT arrays, such as the mitotic spindle or neuronal MT networks, are built and utilized to ensure proper cellular logistics is a central challenge in cell biology.
Before the start of this project, our lab has introduced a new technique, motor-PAINT, to directly resolve MT polarity and the relation between MT orientations, stability and modifications. This revealed that in neurons, the mixed polarity MT network in the dendrites is much more ordered than previously anticipated. MTs with opposite orientations have different properties and are preferred by distinct kinesins, revealing an architectural principle that could explain why different plus-end directed motors move towards distinct destinations. Nevertheless, the mechanisms by which this specialized organization is established and the different ways in which it modulates intracellular transport have remained unknown.
To resolve how cytoskeletal organization guides transport, this project studies the form, formation and functioning of the neuronal MT cytoskeleton. We combine advanced microscopy, molecular biology, and mathematical modelling to: 1) Create a complete 3D map of the dendritic MT cytoskeleton – form. 2) Unravel the mechanisms that establish MT organization in dendrites – formation. 3) Explore how specific MT configurations modulate intracellular transport – function.
Through this research, we are uncovering key mechanisms of cytoskeletal organization and transport in neurons. In addition, our techniques and concepts will aid understanding intracellular transport in other cellular systems. Although most of our work is fundamental research into the mechanisms by which complex cells organize their intracellular logistics, this research is important for society because it provide new tools and concepts to explore and understand basic cell biology. Since most diseases emerge because cells go awry, a deep understanding of cell biology is essential to understand disease and design proper intervention strategies.

We have devoted much of our time to developing better tools to study the nanoscale architecture of the neuronal microtubule cytoskeleton. By combining different super-resolution techniques, such as STED and Expansion Microscopy, we were able to reveals that, in dendrites, stable microtubule enrich at the core of the dendritic shaft, surrounded by a shell of dynamic microtubules. In addition, we established an analysis pipeline that enabled us to estimate the absolute number of dynamics and stable microtubules. We have also developed improved protocols for expansion microscopy of the neuronal cytoskeleton, including a robust protocol for tenfold expansion.

Next to these super-resolution methods, we have established a live-cell marker for stable microtubules and improved optogenetic tools for controlled organelle transport.

In addition to these technical achievements, we have explored the interplay between microtubule dynamics and polarized transport during neuronal development and polarization. This revealed an unanticipated flow of the microtubule network that instructed local transport, indicating that the interplay between microtubule organization and transport is even more complex than we anticipated.

Progress beyond state of the art:

1. Discovery of the radial ordering of stable and dynamic microtubules within mature dendrites ( published in Katrukha et al. eLife 2021)
2. Discovery of the relation between microtubule flow and polarized transport in developing neurons ( preprinted in Burute et al. BioRxiv 2021)
3. Development of Tenfold Robust Expansion Microscopy - TREx – Following our preprint (Damstra et al., BioRxiv 2021), this methods has found its way into many research labs and resulted in various publications
4. Development of an improved toolbox for optogenetics (Nijenhuis et al. JCB 2020)
5. Development of a live-cell marker for stable microtubules (preprinted in Jansen et al, BioRxiv 2021)

Expected results:

- map of the dendritic microtubule array during different stages of development
- mechanistic insights into the mechanisms that shape the dendritic microtubule array
- mechanistic insights into how dendritic microtubule organization controls intracellular transport

Periodic Reporting for period 3 - CellularLogistics (Cellular Logistics: Form, Formation and Function of the Neuronal Microtubule Cytoskeleton)

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