Kinetochore protein Spindly controls microtubule polarity in Drosophila axons
Posted on: 27 April 2020 , updated on: 12 May 2020
Preprint posted on 20 March 2020
Article now published in Proceedings of the National Academy of Sciences at http://dx.doi.org/10.1073/pnas.2005394117
Spindly, a dynein adaptor at the kinetochore, takes on a new and exciting role in the postmitotic neuron: it is required for the determination of axonal microtubule polarity by recruiting dynein to cortical actin.
Selected by Mafalda Pimentel
Categories: cell biology, developmental biology, neuroscience
Background
Neurons are highly specialized cells: they are stimulated at the dendrites, the signal passes through the cell body, and the information is conveyed along a single axon to downstream cells. Due to this extreme polarization, neurons constitute an excellent model to study microtubules and motor- dependent transport. Importantly, the axon has a unique microtubule arrangement, with the growing plus ends oriented away from the cell body and towards the tip. The authors of this preprint have previously shown that in order for this orientation to materialize, the microtubules of the opposite orientation have to be excluded from the axon (1). This sorting is performed by the dynein/dynactin motor complex, which travels on microtubules towards their minus end. Furthermore, dynein must be anchored to the cortical actin in the axon membrane for microtubules to slide (Figure 1). How dynein and actin are connected was unknown.
Figure 1 – In the axon, anchoring of dynein to the cortical actin slides minus-end-out microtubules towards the cell body, and plus-end-out microtubules towards the axon tip. Adapted from reference 1, U. del Castillo et al. (2015) eLife.
Key findings
In this work, the authors aimed to find the adaptor protein responsible for anchoring dynein at the axon cortex.
To achieve this, they performed an RNAi screen using an interesting system: Drosophila S2 cells treated with cytochalasin D (actin severing drug) to generate microtubule-filled membrane projections (2). These projections resemble neuronal processes – the axon in particular – since they display the microtubule same orientation (plus-end-out). The authors used this system previously to demonstrate that the binding of dynein to actin causes this microtubule polarization (1).
From their screen of 22 candidates, only Spindly knockdown lead to a striking mCherry-CAMSAP3 (minus-end binder) accumulation at the processes’ tips. This mixed microtubule orientation phenotype, similar to when Dynein is depleted, was also confirmed by following EB1 comets (plus-end binder).
To test if Spindly (a dynein adaptor) is capable of binding to actin, the authors performed in vitro co- pelleting assays and confirmed an interaction with F-actin. They also expressed a full length GFP tagged version of Spindly and observed it co-localizing with cortical actin in S2 cells.
They further confirmed this function of Spindly in Drosophila axons, by depleting it with an shRNA under a neuron-specific driver (elav-Gal4). These experiments were performed in primary neuronal cultures (from third-instar larval brains) and in class IV dendritic arborization (da) sensory neurons in vivo. The phenotype of Spindly RNAi could be rescued in primary cultures by expressing constructs containing a functional N-terminal domain, which is responsible for the dynein/dynactin interaction.
In vivo, the authors found an enrichment of Spindly in the cell body and axons of photoreceptor neurons and da sensory neurons. When they depleted Spindly, it specifically affected axonal patterning of photoreceptor neurons. These elav>Spindly RNAi larvae developed until adulthood, yet the lifespan and motility of the flies was drastically affected.
Why is this preprint important?
The specificity of the dynein/dynactin complex for each of its multiple functions relies on the binding of adaptors and regulators. Spindly is a well know dynein adaptor at the kinetochore, but its relevance beyond mitosis has only recently begun to be explored (3). This work describes for the first time the role of Spindly as a dynein adaptor to F-actin, and its importance for neuronal development in vivo. Not only does this reinforces the fact that an adaptor might have different roles depending on the cellular context, but also that the microtubule and actin cytoskeleton are tightly interdependent. This new dynein-actin bridge might unlock many paths in the cytoskeleton field.
Future directions
It remains to be understood how:
- Spindly gets enriched in the axon and not in dendrites;
- Spindly binds to actin and what other players might be involved.
Questions to the authors
- Given the work of Lukas Kapitein (4) showing a spatial segregation of differently oriented microtubule bundles, would it be possible that anchoring dynein to the cortex favours a plus-end-out enrichment, instead of excluding minus-end-out?
- Could Spindly be locally translated at the developing axon? Do you have temporal data regarding its axonal enrichment?
- Could Spindly-bound dynein have higher preference towards certain microtubule modifications?
- Have you tested if the localization of GFP-SpindlyFL at the cortex of S2 cells is actin dependent? And the enrichment in axon?
References
- U. del Castillo, M. Winding, W. Lu, V. I. Gelfand, Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons. eLife (2015)
- W. Lu, U. Del Castillo, V. I. Gelfand, Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons. JoVE (2013)
- C. Conte, M. A. Baird, M. W. Davidson, E. R. Griffis, Spindly is required for rapid migration of human cells. Biol Open (2018)
- Tas RP, Chazeau A, Cloin BMC, Lambers MLA, Hoogenraad CC, Kapitein LC, Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport. Neuron (2017)
doi: https://doi.org/10.1242/prelights.19460
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