© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs230086. doi:10.1242/jcs.230086 RESEARCH ARTICLE The interaction between CASK and the tumour suppressor Dlg1 regulates mitotic spindle orientation in mammalian epithelia Andrew P. Porter‡, Gavin R. M. White, Natalie A. Mack* and Angeliki Malliri‡ ABSTRACT orientation in symmetrically dividing epithelial cells is less well Oriented cell divisions are important for the formation of normal understood. Epithelial cells tend to divide in the plane of the epithelial structures. Dlg1, a tumour suppressor, is required for mitotic epithelium, which is important to integrate daughter cells within the spindle orientation in Drosophila epithelia and chick neuroepithelia, epithelium and thereby maintain barrier function (Bergstralh et al., but how Dlg1 is localised to the membrane and its importance in 2013a). These oriented divisions are hypothesised to have a tumour- mammalian epithelia are unknown. We show that Dlg1 is required in suppressive function (Pease and Tirnauer, 2011), although direct non-transformed mammalian epithelial cells for oriented cell divisions evidence of its importance for tumourigenesis is limited at this point. and normal lumen formation. We demonstrate that the MAGUK protein A conserved set of proteins has been identified that regulate CASK, a membrane-associated scaffold, is the factor responsible spindle orientation across a range of organisms and tissue types for Dlg1 membrane localisation during spindle orientation, thereby by defining membrane domains for the attachment of astral identifying a new cellular function for CASK. Depletion of CASK leads microtubules, which in turn orient the mitotic spindle (di Pietro α to misoriented divisions in 3D, and to the formation of multilumen et al., 2016). These include G i, which is involved in the localisation structures in cultured kidney and breast epithelial cells. Blocking of leucine-glycine-asparagine (LGN, also known as GPSM2) and the CASK–Dlg1 interaction with an interfering peptide, or by deletion of subsequently nuclear and mitotic apparatus (NuMA, also known as the CASK-interaction domain of Dlg1, disrupts spindle orientation NUMA1) proteins to the plasma membrane (reviewed in Lu and and causes multilumen formation. We show that the CASK–Dlg1 Johnston, 2013). In turn, NuMA binds to and stabilises astral interaction is important for localisation of the canonical LGN–NuMA microtubules, and recruits the microtubule-binding motor protein complex known to be required for spindle orientation. These results dynein (Du et al., 2002). In mammalian cells, pulling forces at the cell establish the importance of the CASK–Dlg1 interaction in oriented cell cortex are transmitted along astral microtubules which then act to division and epithelial integrity. correctly position the mitotic spindle (Bergstralh et al., 2017). In planar divisions of HeLa cells on L-shaped micropatterns, This article has an associated First Person interview with the first and in chick neuroepithelial cells, LGN localisation is regulated author of the paper. in part by interaction with discs large homolog 1 (Dlg1); LGN membrane localisation is reduced following Dlg1 depletion KEY WORDS: CASK, Dlg1, Mitosis, Mitotic spindle orientation, (Saadaoui et al., 2014), while in Drosophila follicular epithelia Oriented cell division, Lumen formation Dlg1 loss leads to redistribution of Pins (the Drosophila orthologue of LGN) (Bergstralh et al., 2013b). However, in other systems, INTRODUCTION interaction with E-cadherin is required for localisation of LGN Control of the orientation of cell division – via correct positioning of (Gloerich et al., 2017). Whether Dlg1 plays a role in orienting the the mitotic spindle – plays an important role in developing and mitotic spindle along the apical–basal axis in non-transformed maintaining tissue architecture in both embryonic and adult tissues mammalian epithelial cells has not been determined, and the factor (Bergstralh et al., 2017). Oriented divisions have been observed in a regulating Dlg1 membrane localisation in the context of spindle diverse range of organisms and cell types, including yeast, the fruit orientation has yet to be identified (Bergstralh et al., 2017). fly Drosophila, chick neuroepithelial and mammalian epithelial In this report we show that Dlg1 is required for spindle orientation systems, and occur in both stem cells and differentiated tissues (di in 3D cultures of untransformed mammalian epithelial cells, and Pietro et al., 2016). Regulated changes in spindle orientation can identify the membrane-associated guanylate kinase (MAGUK) control the balance between proliferation and differentiation in protein CASK as the protein responsible for Dlg1 membrane stratified epithelia (Bergstralh et al., 2013a). The role of spindle localisation in the context of spindle orientation. By blocking CASK–Dlg1 binding we show that this protein–protein interaction is required for Dlg1 localisation, and subsequently the localisation Cell Signalling Group, Cancer Research UK Manchester Institute, The University – of Manchester, Alderley Park, Macclesfield SK10 4TG, UK. of the LGN NuMA complex, which binds the astral microtubules *Present address: School of Biosciences, University of Nottingham, Sutton that ultimately orient the mitotic spindle. We also show that Bonington Campus, Loughborough, LE12 5RD, UK. blocking the CASK–Dlg1 interaction leads to the formation of ‡Authors for correspondence ([email protected]; multilumen structures. [email protected]) RESULTS A.P.P., 0000-0002-3353-7002; A.M., 0000-0001-6848-090X Dlg1 regulates spindle orientation and epithelial lumen This is an Open Access article distributed under the terms of the Creative Commons Attribution formation in mammalian cells License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. MDCKII cells seeded onto Matrigel have the capacity to grow as cysts, reminiscent of those found in the mammalian kidney, with a Received 18 January 2019; Accepted 14 June 2019 hollow lumen surrounded by a single layer of epithelial cells. We Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230086. doi:10.1242/jcs.230086 Fig. 1. Dlg1 regulates epithelial lumen formation and mitotic spindle orientation. (A) Western blot showing depletion of Dlg1 following transfection with two distinct siRNAs. (B) Confocal images of MDCKII cysts transfected with non-targeting siRNA (siControl), or siRNA targeting Dlg1 (siKD#1 and siKD#2), grown in 2% Matrigel, showing multilumen structures, marked with strong actin staining, following Dlg1 depletion. (C) Quantification of single-lumen cysts from three independent Dlg1 knockdown experiments, n>40 cysts per experiment; ****P<0.0001 (one-way ANOVA). (D) Confocal images of MDCKII cysts grown in collagen I, constitutively expressing non-targeting shRNA (shControl) or shRNA targeting Dlg1 (Dlg1 shKD#1), stained for the indicated proteins to show lumens (upper panels) or Dlg1 levels (lower panels; heatmap of expression levels, arbitrary units), showing multilumen structures after Dlg1 depletion. (E) Quantification of single-lumen cysts from three independent Dlg1 knockdown experiments as in D, n>100 cysts per experiment; **P=0.0083 (unpaired Student’s t-test, two-tailed). (F) Schematic showing measurement of spindle orientation in MDCKII cysts, showing a normally oriented division (>80°) and a misoriented division (<30°). (G) Representative confocal images of MDCKII cysts grown in collagen I and expressing shControl or Dlg1 shKD#1. Mitotic cells are magnified in inserts (lower panels) and annotated to show mitotic spindle angles. (H) Quantification of spindle angles in constitutive knockdown experiments as in G, n=52 and 47 cells pooled from three independent experiments; ***P=0.0004. Data analysed using Mann–Whitney test. (I) Images of early cell divisions (metaphase, left panels; telophase, right panels) in developing MDCKII cysts grown in collagen I. Shown are representative images from shControl and Dlg1 shKD#1 cells, showing misoriented divisions following Dlg1 depletion. (J) Quantification of early cell divisions in MDCKII cysts from experiments as in I, n=95 and 78 from three independent experiments; ***P<0.0001 (Mann–Whitney test). Error bars show mean±s.e.m. Scale bars: 10 µm. knocked down Dlg1 using two independent siRNAs (Fig. 1A) and cells embedded in a pure collagen I matrix; the cells are entirely saw that this disrupted normal lumen formation in Matrigel 3D surrounded by collagen and so have no external polarity cues, unlike culture, giving rise to cysts with multiple lumens, as marked by Matrigel culture where they are seeded on a layer of Matrigel under strong apical actin staining (Fig. 1B, quantified in C). We next grew an upper layer of media supplemented with 2% Matrigel. Single Journal of Cell Science 2 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230086. doi:10.1242/jcs.230086 MDCKII cells grown for eight to 10 days embedded in this mental dysfunction and craniofacial abnormalities in humans anisotropic collagen I matrix produce cysts with a single lumen, as (Hackett et al., 2010). It is well-characterised in neurons (Hsueh, marked with apical actin and GP135 (podocalyxin) staining 2006) where it regulates both trafficking (Lin et al., 2013) and (Fig. 1D, top left panel).