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Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

Masako Okumura, Toyoaki Natsume, Masato T Kanemaki, Tomomi Kiyomitsu

Preprint posted on March 06, 2018 https://www.biorxiv.org/content/early/2018/03/06/277202

Let there be light! Shining light on spindle pulling forces using optogenetics.

Selected by Arnaud Monnard

Context

In this preprint, Okumura et al. used an optogenetic tool to target NuMA (nuclear mitotic apparatus, a protein required for proper mitotic spindle orientation) to subcortical structures. They chose to use the recently published tool – improved Light Inducible Dimer (iLID)1. Interest in and use of cellular optogenetic tools has increased in recent years and although the design of optogenetic tools might differ, their working principles are quite similar2. They largely consist of two protein binding partners in which the interaction between the proteins has been engineered such that they can only bind when exposed to a particular wavelength of light2.

In this preprint, the authors linked one such binding partner to the cell membrane while tagging NuMA-derived constructs and dynein with the second binding partner. In doing so they created photorecruitable versions of NuMA, which they were then able to recruit to subcortical regions using blue light (488 nm).

Additionally, one of the keys to achieving subcellular localization of a protein is successful disruption of the protein’s endogenous localization while minimizing the potential detrimental effects of this disruption. In this preprint, Okumura et al. successfully achieved this by using LGN RNAi, which disrupted NuMA endogenous localization and allowed them to specifically target it to subcortical regions using blue light.

The Preprint

The authors study NuMA (Mud in flies) spatiotemporal requirements to generate cortical pulling forces. During mitosis, correct spindle orientation and positioning ensures accurate chromosomal segregation3. NuMA plays a crucial role in spindle orientation, a key process in symmetric and asymmetric dividing cells. For example, in asymmetrically dividing Drosophila neuroblasts, spindle orientation in mudmutants is compromised, while the apical-basal polarity axis is maintained4. Using cellular optogenetics and triple knock-in cell lines the authors were able to specifically target endogenous NuMA and dynein to subcortical domains. They initially found that targeting NuMA to the cortex is sufficient to generate pulling forces on microtubules. Subsequently, they showed that targeting cortical dynein alone to the cortex is not sufficient to generate pulling forces.

Adapted from Okumura et al. with permission. Final model showing recruitment and assembly of dynein-dynactin-NuMA (DDN) complex

Following these results, the authors decided to conduct a truncation analysis on NuMA to elucidate the precise role and spatiotemporal requirements of the protein’s domains in generating pulling forces. They found that NuMA recruits dynein dynactin via its N terminus and that the C terminus is required for pulling and proper spindle orientation. Together, these results demonstrate the precise spatiotemporal requirements of NuMA and the fact that NuMA is sufficient to orient the spindle and generate pulling forces.

 

Why I chose this preprint

I was attracted to this preprint based on the elegant technical approach that the authors used to elucidate the biological function of dynein-dynactin-NuMA complexes. I was particularly enthusiastic about the endogenous tagging of NuMA, thus minimizing additional cellular perturbations. The growing use of optogenetic tools combined with live-cell imaging will help us to extend our knowledge of dynamic biological processes, while maintaining physiological conditions as much as possible. Moreover, the expansion of this technology to cells in their native environment – such as in intact organisms and intact tissues – offers novel prospects for cell and molecular biology.

 

Questions for the authors

Using your triple knock-in cell lines, have you been able to or tried to create asymmetric cell division?

If so, do you observe a difference in cell fate between the artificially created small and large cell?

 

References

  1. Guntas, G.et al.Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci.112,112–117 (2015).
  2. Guglielmi, G., Falk, H. J. & De Renzis, S. Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis. Futur. Cell Biol.4,1–11 (2016).
  3. di Pietro, F., Echard, A. & Morin, X. Regulation of mitotic spindle orientation: an integrated view. EMBO Rep.17,1106–30 (2016).
  4. Siller, K. H., Cabernard, C. & Doe, C. Q. The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nat. Cell Biol.8,594–600 (2006).

 

The Welburn lab have also highlighted this preprint, check out their post here 

 

Tags: optogenetics

Posted on: 15th May 2018

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  • Author's response

    Tomomi Kiyomitsu shared

    Using your triple knock-in cell lines, have you been able to or tried to create asymmetric cell division?
    I have tried, but I could create only slightly unequal-sized daughter cells. I found that asymmetric membrane elongation partially rectifies light-induced spindle displacement during anaphase.

    Thus, it might be important to control both cortical spindle-pulling forces and membrane elongation for generating extremely unequal-sized daughter cells in symmetrically dividing human cells.

     

    If so, do you observe a difference in cell fate between the artificially created small and large cell?

    If I could, I would love to analyze a difference in cell fate between daughters.

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