Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Urko del Castillo, Hans-Arno J. Müller, Vladimir I. Gelfand

Preprint posted on 20 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.20.000364v1

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)

Tags: actin, axon, cytoskeleton, dynein, polarity, spindly

Posted on: 27 April 2020 , updated on: 12 May 2020

doi: https://doi.org/10.1242/prelights.19460

Read preprint

(1 votes)

Author's response

The authors shared

1) Indeed, both functions of cortical dynein, exclusion of minus-end-out and enrichment of plus-end-out microtubules are compatible and both are likely required. The direction by which cortical dynein moves microtubules is defined by the intrinsic polarity of the microtubules. If a microtubule is oriented correctly (plus-end-out) it will be transported towards the tip and thus enriched of the axons. In contrast, minus-end-out microtubules will be transport towards the cell body and thus excluded from axons (both functions are illustrated in fig 1).

2) It certainly is possible, but currently we do not have any experimental data supporting it.

3) We cannot discard the possibility that cortical dynein displays a preference for a set of microtubules, e.g. those with a particular post-translation modification. However, we favour a scenario where microtubule length and/or microtubule-bundling complexity may be important factors that can affect the efficiency of the microtubule transport driven by spindly/dynein complex.

4) Yes, actin depolymerization with high concentration of LatB prevents cortical enrichment of Spindly in S2 cells. However, we have not tested that in neurons. We predict that depolymerization of cortical actin will affect the localization of Spindly in neurons as well.

Have your say Cancel reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Also in the cell biology category:

CTCF is essential for proper mitotic spindle structure and anaphase segregation

Katherine Chiu, Yasmin Berrada, Nebiyat Eskndir, et al.

Selected by Saanjbati Adhikari

Actin nucleators safeguard replication forks by limiting nascent strand degradation

Jadwiga Nieminuszczy, Peter R. Martin, Ronan Broderick, et al.

Selected by Roberto Amadio

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis

Daniel Salas-Lloret, Néstor García-Rodríguez, Lisanne Giebel, et al.

Selected by Pierre Caron

Also in the developmental biology category:

Coordinated growth of linked epithelia is mediated by the Hippo pathway

Sophia Friesen, Iswar K. Hariharan

Selected by Girish Kale

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila

Archan Chakraborty, Nora G. Peterson, Juliet S. King, et al.

Selected by Anastasia Moraiti

Unclearing Microscopy

Ons M’Saad, Michael Shribak, Joerg Bewersdorf

Selected by Robert Mahen

Also in the neuroscience category:

Alumni

marmite defines a novel conserved neuropeptide family mediating nutritional homeostasis

AP Francisco, I Tastekin, AB Fernandes, et al.

Selected by Pablo Ranea Robles, Cláudia Gil

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Julia Kolb, Nora John, Kyoohyun Kim, et al.

Selected by Laura Celotto

Functional synapses between small cell lung cancer and glutamatergic neurons

Anna Schmitt, Vignesh Sakthivelu, Kristiano Ndoci, et al.

Selected by Jade Chan

preLists in the cell biology category:

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Background

Key findings

Why is this preprint important?

Future directions

Questions to the authors

References

Share this:

Have your say Cancel reply

Sign up to customise the site to your preferences and to receive alerts

Also in the cell biology category:

CTCF is essential for proper mitotic spindle structure and anaphase segregation

Actin nucleators safeguard replication forks by limiting nascent strand degradation

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis

Also in the developmental biology category:

Coordinated growth of linked epithelia is mediated by the Hippo pathway

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila

Unclearing Microscopy

Also in the neuroscience category:

marmite defines a novel conserved neuropeptide family mediating nutritional homeostasis

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Functional synapses between small cell lung cancer and glutamatergic neurons

preLists in the cell biology category:

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

FENS 2020

Planar Cell Polarity – PCP

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

Cell Polarity

TAGC 2020

3D Gastruloids

ECFG15 – Fungal biology

ASCB EMBO Annual Meeting 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Autophagy

Lung Disease and Regeneration

Cellular metabolism

BSCB/BSDB Annual Meeting 2019

MitoList

Biophysical Society Annual Meeting 2019

ASCB/EMBO Annual Meeting 2018

Also in the developmental biology category:

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

2nd Conference of the Visegrád Group Society for Developmental Biology

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

EMBL Conference: From functional genomics to systems biology

Single Cell Biology 2020

Society for Developmental Biology 79th Annual Meeting

FENS 2020

Planar Cell Polarity – PCP

Cell Polarity

TAGC 2020

3D Gastruloids

ASCB EMBO Annual Meeting 2019

EDBC Alicante 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

SDB 78th Annual Meeting 2019

Lung Disease and Regeneration

Young Embryologist Network Conference 2019

Pattern formation during development

BSCB/BSDB Annual Meeting 2019

Zebrafish immunology

Also in the neuroscience category:

FENS 2020

ASCB EMBO Annual Meeting 2019

SDB 78th Annual Meeting 2019

Autophagy

Young Embryologist Network Conference 2019