Kinetochore protein Spindly controls microtubule polarity inDrosophilaaxons

Abstract

24Microtubule polarity in axons and dendrites defines the direction of intracellular transport 25 in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends 26 facing the tips of the processes (plus-end-out), while dendrites contain microtubules with 27 minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical 28 actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, 29 a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor 30 required for dynein-dependent microtubule sorting in axons of Drosophila neurons. 31Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal 32cultures. In addition to these defects, depletion of Spindly in neurons causes major 33 collapse of axonal patterning in the third-instar larval brain as well as severe coordination 34 impairment in adult flies. These defects can be fully rescued by full-length Spindly, but 35 not by variants with mutations in its dynein-binding site. Biochemical analysis 36 demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between 37 dynein and cortical actin in axons. Therefore, Spindly plays a critical role during 38 neurodevelopment by mediating dynein-driven sorting of axonal microtubules. 39 40 Significance Statement 41Neurons send and receive electrical signals through long microtubule-filled neurites called 42 axons and dendrites. One of the main structural differences between axons and dendrites 43 is how their microtubules are organized. Axons contains microtubules with their plus-ends 44 out while microtubules in dendrites are organized with mixed or plus-end-in orientation. 45Dynein, the main minus-end microtubule motor, anchored to cortical actin filaments in the 46 axons is responsible for the uniform microtubule polarity in axons. However, it is unknown 47 how dynein is recruited to the actin cortex in axons. The major finding of this work is that 48Spindly, a protein involved in anchoring dynein to kinetochores during cell division, has a 49 second important function in interphase cells recruiting dynein to the actin cortex in axons. 50 51 Introduction 52Neurons are post-mitotic cells that transmit electrical signals through long neurites called 53 axons and dendrites. Electric signals captured by dendrites are sent unidirectionally 54 through axons and transmitted to other cells. The polarity of microtubules is strikingly 55 different in these two types of neuronal processes. Axons contain microtubules oriented 56 predominantly with their plus-ends-out, while dendrites contain a large fraction of minus-57 end-out microtubules (1, 2). Failure in establishing correct polarity of microtubules results 58 in defects of cargo sorting (3). The differences in microtubule orientation between axons 59 and dendrites are gradually established during development. In cultured early stage 60 neurons, non-polarized neurites contain microtubules with mixed orientation. Later the 61...

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

“…Spindly has been mainly studied as a mitotic adaptor of Dynein-Dynactin (DD), but recent observations suggest that its role might extend to interphase cells (Del Castillo et al, 2020). Here, we have dissected a mechanism of conformational control that addresses the functions of Spindly at kinetochores, and that might inform future studies of Spindly in other cellular locales and cell cycle phases.…”

Ataxin-2 is essential for cytoskeletal dynamics and neurodevelopment in Drosophila

“…Therefore, we investigated the possibility that microtubule dynamics may be altered upon Atx2 knockdown. Initially, we chose to use Drosophila S2 cells to test this hypothesis as they are an efficient system to study the cytoskeleton using dsRNA strategies (22, 23). We depleted Atx2 in Drosophila S2 cells and carried out immunostaining for acetylated tubulin.…”

“…For example, Atx2 has been previously indicated in actin regulation in the germline (Satterfield et al, 2002) and microtubule organization in mitosis (Gnazzo et al, 2016;Stubenvoll et al, 2016). Collectively, these observations are of note because there are strong parallels between organization of the cytoskeleton in mitosis and in neurons (Del Castillo et al, 2020;Cheerambathur et al, 2019;Hertzler et al, 2020;Lin et al, 2012;Norkett et al, 2020;Zhao et al, 2019). In mitosis, the cytoskeleton must be reorganized to facilitate chromosome separation and actin ring contraction (Basant and Glotzer, 2018;Mishima et al, 2002).…”

Ataxin-2 is essential for cytoskeletal dynamics and neurodevelopment in Drosophila

“…For example, Atx2 has been previously indicated in actin regulation in the germline (Satterfield et al, 2002) and microtubule organization in mitosis (Gnazzo et al, 2016;Stubenvoll et al, 2016). Collectively, these observations are of note because there are strong parallels between the organization of the cytoskeleton in mitosis and in neurons (Del Castillo et al, 2020;Cheerambathur et al, 2019;Hertzler et al, 2020;Lin et al, 2012;Norkett et al, 2020;Zhao et al, 2019). In mitosis, the cytoskeleton must be reorganized to facilitate chromosome separation and actin ring contraction (Basant and Glotzer, 2018;Mishima et al, 2002).…”