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

Project Information

CellularLogistics

Grant agreement ID: 819219

DOI

10.3030/819219

Project closed

EC signature date 18 February 2019

Start date 1 May 2019

End date 30 April 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 4 - CellularLogistics (Cellular Logistics: Form, Formation and Function of the Neuronal Microtubule Cytoskeleton)

Reporting period: 2023-11-01 to 2024-04-30

Summary of the context and overall objectives of the project

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 has studied the form, formation and functioning of the neuronal MT cytoskeleton. We have combined 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 have uncovered key mechanisms of cytoskeletal organization and transport in neurons. In addition, the techniques and concepts developed in this project are now also applied to 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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In key objective 1, we have development many new tools in order to better map MT organization in dendrites. This resulted in improved protocols for Expansion Microscopy (Damstra et al., eLife 2022, Damstra et al., Nature Methods 2023) and for 3D motor-PAINT (Iwanski et al., Methods Mol Biol 2024). In addition, we have used these technologies to obtain quantitative information about the spatial distribution and abundance of the two key subsets on MT in dendrites, stable and dynamic MTs (Katrukha et al., eLife 2021)

In key objective 2, we have studied the mechanisms by which the dendritic MT array is established. For this we used motor-PAINT to study changes in MT polarity during different stages of development. This revealed that stable MTs undergo polarity reversal during stage 2. To map this transition in live-cells, we first developed and validated a new tools that allows us to selectively visualize stable microtubules and observed their dynamics (Jansen et al., Journal of Cell Biology 2023). Using this tools we could directly visualize the polarity reversal of stable microtubules and identify different mechanism through which this occurs. The manuscript about this is currently in preparation. Finally, we have used modelling to explore how different mechanisms can lead to symmetry breaking and polarity reversal during early neuronal development.

In key objective 3, we have explored how specific MT configurations modulate intracellular transport. For example, we have studied how the selective accumulation of Kinesin-1 in single neurites depends on the organization of the MT network. We discovered that the MT network in most neurites undergoes slow retrograde movement in most neurites, except in the one where Kinesin-1 accumulates. Here the network more anterograde or is immobile. To test the causal relationship between these events, we have developed tools that allowed us to acutely immobilize the MT network in either all or individual neurites. This revealed that MT immobilization (by anchoring to the plasma membrane) is sufficient to trigger selective accumulation of Kinesin-1 (Burute et al, Science Advances 2022). Also for this key objective, modeling has helped us to better understand these phenomena and we are currently still working on a theoretical manuscript.

In summary, our research has uncovered key mechanisms of cytoskeletal organization and transport in neurons. The key insights that emerged from this work have been presented at many scientific meetings and published in open-access peer-reviewed journals. Many of the tools and approaches that we have pioneered are now also used by other groups.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

New insights into the neuronal cytoskeleton:
1. Quantitative information on the distribution of different subsets (Katrukha et al, eLife 2021)
2. First live-cell marker for stable microtubules (Jansen et al, Journal of Cell Biology, directly revealing polarity reversal (Iwanski et al, in preparation)
3. New insight into the development of the neuronal microtubule network (Burute et al, Science Advances 2022)

Tool development for expansion microscopy
4. GelMap patent application and setting up spin-off company
5. Collaborations with pathology and industry

In my opinion, achievements 1-3 all advance the research field beyond the state-of-the-art and have enabled complete new insights into the development of the neuronal MT cytoskeleton. These developments were mostly along the lines of the research plan and therefore not entirely unexpected. Achievements 4 and 5, on the other hand, were completely unplanned and followed from our development of the first intrinsic deformation mapping method for expansion microscopy.

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