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GAS2-Like 1 Coordinates Cell Division Through Its Association with End www.nature.com/scientificreports OPEN GAS2-like 1 coordinates cell division through its association with end-binding proteins Received: 30 November 2018 Alicja Nazgiewicz, Paul Atherton & Christoph Ballestrem Accepted: 15 March 2019 Cell division involves the tightly coordinated rearrangement of actin and microtubules (MTs). We have Published: xx xx xxxx previously shown that a member of the family of growth arrest-specifc 2-like proteins, GAS2-like 1 (G2L1) regulates actin-MT crosstalk through its associations with plus-end microtubule tip-binding (EB) proteins. Here we show that G2L1 is involved in the regulation of cell division. We show that the depletion of G2L1 results in a reduction in the number of cells undergoing cell division and a signifcant proportion of those cells that do divide are either multinucleated, display deformed nuclei, or undergo cell division at a much slower rate. Exogenous expression of G2L1 mutants revealed that the association of G2L1 with EB1 is critical for regulated cell division and blocking this interaction inhibits cell division as observed in cells lacking G2L1. Taken together, our data suggest that G2L1 controls the precise regulation and successful progression of cell division through its binding to EB-proteins. Cell division is a vital process in the lifetime of a cell. Any aberrations during this process can lead to severe health problems, and uncontrolled division is a key hallmark of cancer. Coordinated cell division requires precise rearrangements of the actin and microtubule (MT) cytoskeletal systems and the interplay between these two systems is vital. As cells round up prior to mitosis, actin stress fbres (SFs) disassemble into a cortical actomyosin network at the cell periphery. At the same time, MTs reorganise to form the mitotic spindle which is composed of1: kinetochore MTs linked to kinetochores on sister chromatids responsible for chromosome segregation2, non-kinetochore MTs that interact with the same MT type from the opposite spindle pole, and3 astral MTs that attach the spindle to the cell cortex. At the end of anaphase, dividing cells form an actin-based contractile ring, which forms the cleavage furrow during telophase. Components of the cleavage furrow are essential for separa- tion into two daughter cells during cytokinesis (reviewed in)4. A number of reports have shown that the synchronization of mitotic events requires the coordination of both actin and MT networks. For example, it was found that the cortical actin network plays an important role in spin- dle assembly, positioning, and length during mitosis5–7. Te precise mechanism of how the actin-MT interplay is regulated at diferent stages of cell division remains to be elucidated. Te GAS2 protein family consists of four members: the founding member of the family GAS28, and three GAS2-like (G2L) proteins (GAS2-like 1 (G2L1), GAS2-like 2 (G2L2) and GAS2-like 3 (G2L3)), all of which have previously been shown to contribute to cytoskeletal regulation9,10. All members contain an actin-binding calponin homology (CH) domain and a putative MT-binding GAS2-related (GAR) domain. G2L1 and G2L2 con- tain a larger unstructured C-terminus domain with evolutionarily-conserved MT-tip localisation signals (MtLS) composed of the amino acid consensus sequence Ser/Tr-X-Ile/Leu-P (SxIP motifs) (Fig. 1A)9. Tis motif is required for the interaction with MT plus-end (+end)-binding (EB) proteins11 and regulate the crosstalk between MTs and F-actin12,13. G2L proteins have previously been shown to contribute to the regulation of cell division. G2L3 knockout mice die early afer birth because of cytokinesis defects14. Depletion of G2L3 resulted in defects in chromosome separa- tion15,16, and overexpression of G2L3 specifcally interferes with cell abscission at the fnal stage of cell division17. In addition to having a role in regulating cell motility12, G2L1 has also been reported to regulate centrosome splitting by mediating actin-microtubule crosstalk1. However, the precise role of G2L1 in cell division requires further elucidation. Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PT, UK. Correspondence and requests for materials should be addressed to C.B. (email: christoph. [email protected]) SCIENTIFIC REPORTS | (2019) 9:5805 | https://doi.org/10.1038/s41598-019-42242-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports SRIP A 1 681 G2L1 CH GAR B G2L1-FL MTs Actin Merge Figure 1. GAS2 family members and their subcellular localisation in U2OS cells. (A) Schematic representation of GAS2-Like1 (G2L1). Te calponin homology (CH) and GAS2-related (GAR) domains are depicted in red and orange, respectively, and the number of amino acids is noted above the C-termini (green). Te IP motives responsible for end-binding proteins (EB) binding are denoted above their respective C-termini, SxIP motif is yellow. (B) U2OS cell expressing GFP-G2L1. Cells were fxed and stained for MTs (blue) and actin (red). White arrowheads indicate co-aligning MTs and actin structures. Scale bar indicates 10 μm. EB proteins have a well-established role in cell division18–21. EB1 has been shown to be involved in anchor- ing MTs at the centrosome22, and has additional roles in regulating spindle positioning and symmetry23,24, the connection of MTs with kinetochores18 and cortical contractility23. Perturbation of EB1-MT association results in failure of chromosomal congression and delayed mitotic events18. EB3, on the other hand, has been shown to be responsible for stabilization of the midbody and focal adhesions (FA), which are required for coordinated spreading of daughter cells during cytokinesis19. Despite these observations it remains unclear how EB-proteins are engaged in these processes that are dependent on efcient cross-communication between actin and MTs. Given the importance of both cytoskeletal systems in regulating mitotic events, and the strong regulatory link between G2L1 and EB proteins, we hypothesised a role for G2L1 in regulating cell division by mediating actin-MT crosstalk through EB proteins. In this study, we show that G2L1 localises to the mitotic spindle and cleavage furrow during mitosis and constriction zones of the midbody during late cytokinesis. We found that depletion of G2L1 leads to nuclear abnormalities and multinucleation therefore inhibiting and/or delaying cell division. Tese abnormalities and defects in cell division also occur upon expression of an EB-binding defcient mutant of G2L1. Our data leads to a model whereby G2L1 mediates actin-MT crosstalk through its binding to EB-proteins, to regulate the coordinated progression through cell division. Results G2L1 localises to both actin- and MT-related mitotic structures during cell division. Exogenous expression of G2L1 induces a strong co-alignment of MTs and actin stress fbres (SFs) in NIH 3T3 fbroblasts13. We aimed to confrm this fnding in a human osteosarcoma cell line (U2OS) that is well established for cell division studies. As previously shown for fbroblasts, we detected a strong MT-actin co-alignment for cells exog- enously expressing G2L1 (Fig. 1B). To investigate the potential role of G2L1 in cell division we expressed wild-type G2L1 (G2L1-FL-GFP) in U2OS cells and analysed its localisation at diferent phases of mitosis. To visualise its relation with the cytoskel- etal systems we co-labelled cells for actin, MTs and DNA. Using this approach we identifed G2L1 colocalisation with actin structures including the contractile ring, the cleavage furrow and constriction zones of the midbody. G2L1 also localised to MT-related structures such as the centrosome and the mitotic spindle during cell division (Fig. 2A,B, Supplemental Movie 1). While exogenous G2L1-GFP localised to mitosis-related structures, it remained to be determined whether the endogenous protein shared this localisation. To detect endogenous G2L1 we used an antibody directed against the C-terminal tail of G2L1. To test specifcity and recognition for G2L1, which has very low endogenous expres- sion levels16, we frstly performed western blotting of cells expressing exogenous G2L1-FL-GFP. We found that the antibody detected GFP tagged G2L1 (Supplementary Fig. 1A). Similarly, immunofuorescence (IF) of cells expressing G2L1-FL-GFP using the same antibody demonstrated colocalisation with GFP tagged G2L1 indicating that the antibody was specifc for G2L1 (Supplementary Fig. 1B). We used this antibody to identify the localisation of endogenous G2L1 by IF in U2OS cells. We found faint localisation to MTs in cells in interphase, but observed an accumulation of G2L1 at constriction sites of the mid- body of dividing cells during later stages of cytokinesis (Supplementary Fig. 1B). Tese data suggested a potential role for G2L1 in cell division. G2L1 depletion results in aberrant cell division leading to nuclear abnormalities. To determine the potential role of G2L1 in cell division we depleted the endogenous protein using three diferent G2L1-specifc siRNA. We confrmed siRNA knock down of G2L1 by western blotting (Fig. 3A) and by quantitative PCR (qPCR), which showed a 60–80% reduction of G2L1 mRNA levels compared to control cells (Supplementary Fig. 1C). To visually assess G2L1 knock down in cells, we expressed empty GFP-vector together with siRNA oligos and stained these cells for endogenous G2L1. As endogenous G2L1 was primarily detectable in cells at its accumulation at the midbody during the later stages of
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