ARTICLE https://doi.org/10.1038/s41467-019-10446-z OPEN Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells Benita Wolf1, Coralie Busso1 & Pierre Gönczy1 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. fi 1234567890():,; We develop a live imaging siRNA-based screen using stereotyped bronectin 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 tar- get 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. 1 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland. Correspondence and requests for materials should be addressed to P.G. (email: pierre.gonczy@epfl.ch) NATURE COMMUNICATIONS | (2019) 10:2859 | https://doi.org/10.1038/s41467-019-10446-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10446-z patial control of cell division is crucial during development network24. Furthermore, the tumor suppressor liver kinase B1 and for tissue homeostasis1,2. In animal cells, the position of (LKB1) is needed for proper AMPK distribution and, potentially S 25 the mitotic spindle can dictate the correct segregation of fate thereby, accurate spindle positioning . determinants during cell division, as well as the accurate place- Importantly, spindle positioning is also regulated by cell ment of daughter cells within tissues and organisms. Defective extrinsic cues26 (reviewed in ref. 5). Thus, in non-polarized sys- spindle positioning in the vertebrate central nervous system can tems such as human HeLa cells, the spindle aligns with respect to lead to tissue hyperplasia3, indicative of accurate spindle posi- the substratum via integrin-mediated cell-substrate adhesion. tioning playing a tumor suppressive function4. Overall, proper Besides integrin-linked kinase, such alignment requires astral spindle positioning is critical for the spatial regulation of cell microtubules, the microtubule end-binding protein EB1, cortical division, and an understanding of the underlying mechanisms has dynein, the actin cytoskeleton, and the motor protein myosin important implications for proliferation control. X27–29. Here, cortical actin is thought to link substrate-derived Astral microtubules that emanate from the two poles of the cues to spindle positioning by connecting retraction fibers with mitotic spindle and polymerize toward the cell cortex are key for astral microtubules30. ensuring proper spindle positioning (reviewed in ref. 5). A further Despite significant knowledge regarding the mechanisms gov- critical element is the cortically anchored minus-end directed erning spindle positioning in human cells, it is likely that a more motor dynein, which, together with depolymerizing astral comprehensive understanding could be achieved through the microtubules, generates pulling forces that result in accurate discovery of further components critical for this process. spindle positioning (reviewed in ref. 6). Dynein is anchored at the Accordingly, a live imaging RNA interference (RNAi)-based cell cortex through a ternary complex that comprises notably screen of 107 proteins associated with LGN, Gαi, and dynein/ a large coiled coil protein called NuMA in human cells, LIN-5 in dynactin complexes identified a novel role for the actin capping Caenorhabditis elegans and Mud in Drosophila7–9. In metaphase binding protein Zβ in spindle positioning, a role exerted through human cells, NuMA is anchored at the cell cortex through an an impact on cortical dynein and microtubule dynamics31. interaction with the GoLoco-containing protein LGN, which itself Moreover, a small interfering RNA (siRNA)-based screen of 719 is bound to the plasma membrane through an interaction with kinases and kinase-related components has been performed on aGαi protein8. At this stage of the cell cycle, most of the NuMA fixed cells, thereby capturing endpoint phenotypes19. Although protein is phosphorylated by CDK16,10. Given that this kinase is this screen uncovered notably the role of ABL1 in spindle posi- enriched at centrosomes, phosphorylated NuMa localizes pri- tioning, such an endpoint assay might have missed some key marily at spindle poles during metaphase6. Such phosphorylation components. is counteracted by PPP2CA, which is thought to act throughout Here, therefore, we design and execute a large-scale unbiased the cell, together resulting in a low level of dephosphorylated live imaging functional genomic siRNA-based screen for com- NuMA at the cell cortex6,11. Upon loss of CDK1 activity in ponents mediating spindle positioning in cultured human cells, anaphase, NuMA binds to the cortex independently of LGN screening a set of 1280 genes that comprises notably all kinases through an interaction with phosphatidylinositol 4,5-bispho- and phosphatases. We thus identify 16 components whose inac- 12,13 sphate (PIP2) . Moreover, 4.1 band proteins may contribute to tivation dramatically perturbs spindle positioning, including tyr- NuMA cortical anchoring in anaphase14, although there are osine receptor kinase 3 (TYRO3) and cyclin G-associated kinase contrasting results on this aspect12. (GAK). Follow-up experiments establish that TYRO3 functions Regulation of spindle positioning, in particular through by restricting the extent of cortical NuMA and dynein during alterations of NuMA distribution, relies also on kinases other metaphase, whereas GAK acts by ensuring the presence of robust than CDK1. Thus, Aurora Kinase A phosphorylates NuMA astral microtubules during mitosis. during metaphase, promoting NuMA cortical enrichment and proper spindle positioning15,16. Accordingly, interfering with Aurora A function in the mammary epithelium impairs spindle Results position, resulting in altered cell fate and proliferation17. The Assay development. We set out to design an siRNA-based live Polo-like kinase 2 (PLK2) is also important for accurate spindle imaging functional genomic screen to identify novel modulators orientation in the mammary gland, although whether this is of spindle positioning in human cells. To visualize the position of through the same route as Aurora A is not known18. Another the metaphase plate in time-lapse recordings, we utilized HeLa crucial kinase is the Abelson murine leukemia viral oncogene cells carrying an integrated plasmid expressing mCherry::H2B homolog 1 (ABL1), which phosphorylates NuMA on a distinct (Fig. 1a). Moreover, we took advantage of the ability of the residue than CDK1 or Aurora A, thus ensuring the maintenance substratum to dictate spindle positioning, such that the meta- of cortical NuMA during metaphase19. phase plate of cells grown on L-shaped fibronectin-coated Cell-intrinsic components besides ternary complex members micropatterns is typically positioned with a 45° angle to the arms are also important for proper spindle positioning in human cells. of the L (Fig. 1a)26. This is the case of the acto-myosin network, which ensures cor- As summarized in Fig. 1b and described in more detail in the tical rigidity and thereby prevents the plasma membrane from Methods section, we developed a robust screening pipeline to being pulled inwards by forces exerted by astral microtubules20. identify spindle positioning phenotypes. In brief, HeLa mCherry:: Furthermore, the Ste20-like kinase (SLK) activates ezrin/radixin/ H2B cells were reverse transfected in 96-well plates containing moesin (ERM) proteins, which link cortical actin to the plasma siRNAs directed against genes to be tested, as well as negative membrane, with the loss of SLK or ERM proteins decreasing the controls (ctrl) and positive controls (LGN, which impairs but does amount of LGN and NuMA at the metaphase cortex without not abolish spindle positioning)2 (Fig. 1b). After incubation for influencing Gαi21. Moreover, the unconventional myosin Myo10, 48h, cells were transferred to 96-well imaging plates containing L- as well as the mitotic interactor and substrate of Plk1 (MISP) shaped micropatterns, followed by the imaging of two visual fields protein, is important for proper spindle positioning also through per well once every 8 min during 24h (Supplementary Fig. 1a, b). their ability to couple cortical actin with astral microtubules22,23. To determine spindle position from the resulting recordings, we In addition, phosphorylation of the myosin regulatory light chain used the ImageJ-based pipeline TRACMIT to extract the angle of by AMP-activated protein kinase (AMPK) modulates the asso- the metaphase plate with respect to the arms of the L-shape just ciation between astral microtubules and the cortical acto-myosin before anaphase32 (Fig. 1c, d). 2 NATURE