Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells

Benita Wolf,Pierre Gönczy,Coralie Busso

doi:10.1038/s41467-019-10446-z

Benita Wolf, Pierre Gönczy + Show 1 more

Open Access

https://doi.org/10.1038/s41467-019-10446-z

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Abstract

Proper spindle positioning is crucial for spatial cell division control. Spindle positioning in human cells relies on a ternary complex comprising Gαi1–3, LGN and NuMA, which anchors dynein at the cell cortex, thus enabling pulling forces to be exerted on astral microtubules. We develop a live imaging siRNA-based screen using stereotyped fibronectin micropatterns to uncover components modulating spindle positioning in human cells, testing 1280 genes, including all kinases and phosphatases. We thus discover 16 components whose inactivation dramatically perturbs spindle positioning, including tyrosine receptor kinase 3 (TYRO3) and cyclin G associated kinase (GAK). TYRO3 depletion results in excess NuMA and dynein at the cortex during metaphase, similar to the effect of blocking the TYRO3 downstream target phosphatidylinositol 3-kinase (PI3K). Furthermore, depletion of GAK leads to impaired astral microtubules, similar to the effect of downregulating the GAK-interactor Clathrin. Overall, our work uncovers components and mechanisms governing spindle positioning in human cells.

Highlights

Proper spindle positioning is crucial for spatial cell division control
Astral microtubules that emanate from the two poles of the mitotic spindle and polymerize toward the cell cortex are key for ensuring proper spindle positioning
Polo-like kinase 2 (PLK2) is important for accurate spindle orientation in the mammary gland, whether this is through the same route as Aurora A is not known[18]

Summary

Introduction

Spindle positioning in human cells relies on a ternary complex comprising Gαi[1,2,3], LGN and NuMA, which anchors dynein at the cell cortex, enabling pulling forces to be exerted on astral microtubules. At this stage of the cell cycle, most of the NuMA protein is phosphorylated by CDK16,10 Given that this kinase is enriched at centrosomes, phosphorylated NuMa localizes primarily at spindle poles during metaphase[6]. Polo-like kinase 2 (PLK2) is important for accurate spindle orientation in the mammary gland, whether this is through the same route as Aurora A is not known[18] Another crucial kinase is the Abelson murine leukemia viral oncogene homolog 1 (ABL1), which phosphorylates NuMA on a distinct residue than CDK1 or Aurora A, ensuring the maintenance of cortical NuMA during metaphase[19]

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