19/7/22, 19:31 Repurposing Kinetochore Microtubule Attachment Machinery in Neurodevelopment - ScienceDirect

Developmental Cell
Volume 48, Issue 6, 25 March 2019, Pages 746-748

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Repurposing Kinetochore Microtubule Attachment Machinery

in Neurodevelopment
Rosalind Norkett 1, Wen Lu 1, Vladimir I. Gelfand 1

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https://doi.org/10.1016/j.devcel.2019.03.004 Get rights and content

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Refers to Dhanya K. Cheerambathur, Bram Prevo, Tiffany-Lynn Chow, Neil Hattersley, Shaohe Wang, Zhiling Zhao,
Taekyung Kim, Adina Gerson-Gurwitz, Karen Oegema, Rebecca Green, Arshad Desai

The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Mor…

Developmental Cell, Volume 48, Issue 6, 25 March 2019, Pages 864-872.e7

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Guoli Zhao, Asli Oztan, Yingzhi Ye, Thomas L. Schwarz

Kinetochore Proteins Have a Post-Mitotic Function in Neurodevelopment

Developmental Cell, Volume 48, Issue 6, 25 March 2019, Pages 873-882.e4

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Kinetochore-microtubule attachments are essential to direct proper chromosome segregation

during cell division. In this issue of Developmental Cell, Cheerambathur et al. (2019) and Zhao et al.
(2019) uncover an unexpected role in neuronal development, unrelated to cell division, for
components of the highly conserved kinetochore-microtubule attachment complex.

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19/7/22, 19:31 Repurposing Kinetochore Microtubule Attachment Machinery in Neurodevelopment - ScienceDirect

Main Text
The KMN network is extensively studied with regard to accurate completion of mitosis. This
requires sister chromatids be attached properly to spindle microtubules for the alignment and
segregation of chromosomes to daughter cells. This attachment is achieved by the KMN network,
a highly conserved set of protein complexes named for their major component proteins that
comprised of three complexes: Knl1, Mis12, and Ndc80. This network provides the primary
interface between kinetochores on centromeres and the spindle microtubules. The Knl1 complex
allows recruitment of checkpoint complexes, whereas the Ndc80 complex provides the major
microtubule binding interface, and the Mis12 complex recruits the Knl1 and Ndc80 complexes,
thereby providing a scaffold to connect centromeric DNA to microtubules (Varma and Salmon,
2012). In this issue of Developmental Cell, two parallel studies, Cheerambathur et al. (2019) and
Zhao et al. (2019), respectively, from the Desai and Schwarz groups uncover an unexpected role for
KMN proteins in nervous system development independent of their function in mitosis.

Starting with an unbiased genetic screen in Drosophila embryos to investigate synapse formation,
Zhao et al. (2019) discovered a developmental abnormality at the neuromuscular junction (NMJ)
upon depletion of Mis12. In these embryos, characteristic presynaptic boutons were often absent
(although presynaptic components could still be detected), and neurites were overextended
compared to controls. From these observations, the authors suggest a defect specifically in
extension of neuronal processes rather than a general developmental arrest. Indeed, this
increased neurite outgrowth was also evident in dendrites of sensory neurons in late-stage
embryos, suggesting involvement of this kinetochore component in nervous system development
more generally, not just at the presynapse.

Extending their investigations beyond Mis12, Zhao et al. (2019) considered the impact of reducing
levels of other KMN network proteins. They found increased neurite extension and abnormal
bouton development upon depletion of multiple players in the KMN network including
components of each of the three constituent complexes. Zhao et al. (2019) also described Ndc80
distribution in the neuropil, away from the cell body and putative centromere, and at the NMJ
presynapse. Moreover, the distribution, and that of other KMN proteins, was dependent upon
Mis12. This raises the possibility that these proteins are acting in a comparable network in the
nervous system, and at the NMJ, to the kinetochore.

More direct observations using C. elegans embryos by Cheerambathur et al. (2019) also
demonstrated a role for kinetochore proteins in neurodevelopment. During late embryogenesis,
endogenous GFP-tagged KNL-1 was prominent in the developing head region and showed a
filamentous, potentially microtubule-like distribution. Using split GFP technology in sensory
neurons, the authors confirmed NDC-80 expression in the developing nervous system, post-
mitotically. The authors then used another genetic technique to address the role of kinetochore
proteins in nervous system development—namely, a sensory neuron-specific GFP degrader and
endogenous GFP-tagged KMN proteins. In this system, GFP-tagged proteins are coexpressed with
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a fusion between a GFP-binding nanobody and an ubiquitin ligase adaptor, thus targeting GFP-
containing proteins for degradation. They found that inducing neuron-specific loss of KNL-1 or
NDC-80 caused severe disorganization of the sensory nervous system and increased axon
outgrowth, comparable to observations by Zhao et al. (2019). In contrast, Cheerambathur et al.
(2019) observed decreased dendritic extension upon KNL-1 and NDC-80 degradation.

Given the essential role of the KMN network in kinetochore microtubule attachment (Varma and
Salmon, 2012), Cheerambathur et al. (2019) address this function in neurons, taking advantage of
existing understanding of protein functional domains from chromosome segregation studies.
NDC-80 contains two microtubule binding sites—one within a calponin homology domain,
essential for chromosome segregation, and one in the basic N-terminal tail. GFP-mediated
degradation of endogenous NDC-80 and molecular replacement with mutations/deletions of
both these regions showed abnormal sensory nervous system organization. Notably, these
microtubule binding-deficient NDC-80 mutants did not alter the dendritic localization of other
KMN network components. This perhaps suggests diverging functions of the KMN network
during cell division and in post-mitotic neurons.

Significantly, these findings in invertebrates extend to functions in rodent neurons. Zhao et al.
(2019) show that knockdown of mis12 results in increased “dendritic protrusion” density in
cultured rat hippocampal neurons. This points to conservation of this post-mitotic function of
the KMN machinery from invertebrates to mammals.

Intriguingly, the KMN complex is not the only paradigm of “repurposing mitotic machinery in
neurons.” For example, our group discovered that the mitotic kinesin-6 (Pavarotti) controls
neurite development (Del Castillo et al., 2015). In addition to its canonical role in regulating anti-
parallel microtubule sliding and activating Rho family GTPase, Pavarotti negatively regulates
neurite outgrowth in Drosophila (Del Castillo et al., 2015). Post-mitotic depletion of kinesin-6
caused overextension of motor neuron axons and mistargeting of neuromuscular junctions,
which intriguingly mimic the phenotypes of the mis12 mutant reported by Zhao et al. (2019).
Kinesin-6 controls neurite outgrowth via downregulation of microtubule-microtubule sliding
driven by kinesin-1, which is essential for both axonal and dendritic growth (Lu and Gelfand,
2017, Winding et al., 2016) as well as axon regeneration (Lu et al., 2015). This role in neuronal
cytoskeletal organization is likely not unique to kinesin-6, as kinesin-12, another mitotic motor,
has also been shown to be involved in neuronal development via dendritic determination (Lin
et al., 2012).

Collectively, these studies revise the conventional thinking that so-called “mitotic” proteins
function exclusively in dividing cells, thereby extending the concept of cell division machinery
repurposing in the nervous system. These two new studies raise the questions of how expression
of KMN proteins is developmentally regulated, and how these proteins localize to neuronal
processes. What signaling capacity does the complex have (especially in the context of Knl-1), and
what capacity may these complexes have to organize the microtubule cytoskeleton? How does the
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KMN network coordinate with other repurposed “mitotic” motors? This is of specific interest in
neurons where axons have uniform, plus end-out microtubule polarity, while that in dendrites is
more mixed. Notably, the precise mechanism of loss of KMN proteins leading to altered
outgrowth of neuronal processes remains a mystery—how do kinetochore proteins regulate
neurite morphology?

Recommended articles

References
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Kinetochore proteins have a post-mitotic function in neurodevelopment
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this issue
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