Cytoplasmic Dynein Functions in Planar Polarization of Basal Bodies within Ciliated Cells

Summary Despite common consensus about the importance of planar cell polarity (PCP) proteins in tissue orientation, little is known about the mechanisms used by PCP proteins to promote planar polarization of cytoskeletons within individual cells. One PCP protein Fzd6 asymmetrically localizes to the apical cell membrane of multi-ciliated ependymal cells lining the lateral ventricular (LV) wall on the side that contacts cerebrospinal fluid flow. Individual ependymal cells have planar polarized microtubules that connect ciliary basal bodies (BBs) with the cell cortex of the Fzd side to coordinate cilia orientation. Here, we report that cytoplasmic dynein is anchored to the cell cortex of the Fzd side via an adapter protein Daple that regulates microtubule dynamics. Asymmetric localization of cortical dynein generates a pulling force on dynamic microtubules connected to BBs, which in turn orients BBs toward the Fzd side. This is required for coordinated cilia orientation on the LV wall.


INTRODUCTION
Cilia arise from basal bodies (BBs) docked on apical cell surfaces, and their orientation is determined by planar polarity at the apical cell cortex. Coordinated beatings of multiple motile cilia with coordinated orientation generate directional fluid flow on the tissue surface. This enables brain ventricle circulation of cerebrospinal fluid (CSF), protective airway mucus clearance, and ovarian tube egg transportation (Marshall and Kintner, 2008). Perturbation of coordinated cilia orientation results in hydrocephalus, chronic obstructive pulmonary disease (COPD), and female infertility (Meunier and Azimzadeh, 2016). Ependymal cells that line the lateral ventricular (LV) wall have multiple motile cilia that are oriented toward and beat parallel to the flow of CSF (Mirzadeh et al., 2010;Figures 1A and 1C). In response to the initial passive CSF flow, planar cell polarity (PCP) protein, Vangl2, is required for the rotational orientation of BB/cilia within individual ependymal cells (Guirao et al., 2010). Mutations in other PCP genes (Celsr, Dvl, and Daple) also disrupt BB/cilia orientation and ependymal flow, which results in stagnant CSF and hydrocephalus (Tissir et al., 2010;Boutin et al., 2014;Ohata et al., 2014;Takagishi et al., 2017). As such, PCP proteins are essential for the orientation of ependymal cilia and consequently for the directional active flow of CSF (Guirao et al., 2010;Ohata and Alvarez-Buylla, 2016).
Tissue strain stimulates subcellular asymmetric accumulation of core PCP proteins (Fzd/Dvl and Vangl/Pk) in animals (Carvajal-Gonzalez et al., 2016). PCP proteins are known to organize microtubule polarization, which establishes the planar polarity of cells (Matis et al., 2014;Chien et al., 2015). In tracheal multi-ciliated cells, microtubules show plane polarization at the apical cell cortex and this contributes to BB orientation (Vladar et al., 2012;Kunimoto et al., 2012;Chien et al., 2015). Growing ends of microtubules are localized asymmetrically at the side of the cell cortex, where Fzd accumulates (Vladar et al., 2012;Butler and Wallingford, 2017). However, the mechanisms employed by PCP proteins that are responsible for microtubule organization and cilia orientation remain unclear.
During Wnt signaling, a Wnt ligand binds to the seven-pass transmembrane receptor Fzd, which then recruits a scaffold protein Dvl that induces downstream signaling (Schulte and Bryja, 2007). We have previously reported a loss of microtubule polarization in mice that are deficient for a Dvl-binding protein Daple (Takagishi et al., 2017). In light of these findings, we proceeded to study how Fzd engages growing ends of microtubules and regulates BB orientation. We have demonstrated here that Daple anchors cytoplasmic dynein to the cell cortex of ependymal cells on the LV wall. Cytoplasmic dynein is a cytoskeletal motor protein that traverses microtubules toward the microtubules' minus end that lies within the microtubule-organizing center (Roberts et al., 2013). Cell cortexanchored dynein generates a pulling force on astral microtubules that are connected to the spindle pole during anaphase (Laan et al., 2012). We have found that cytoplasmic dynein is anchored to the Fzd6/ Dvl1/Daple side of the cell cortex and functions in BB positioning and orientation. Our data suggest that cortex-anchored dynein at the Fzd/Dvl/Daple side of the cell cortex generates a pulling force on microtubules connected to BBs and utilizes microtubule dynamics to control positioning and rotation of BBs.

Planar Polarization of Microtubules in Ependymal Cells
Within the LV, CSF flows from the posterior choroid plexus toward the anterior-ventral foramen of Monro ( Figure 1A). Ependymal cells lining LV walls display asymmetric accumulation of PCP proteins, with multiple cilia oriented along the direction of CSF flow (Guirao et al., 2010;Ohata and Alvarez-Buylla, 2016). A core PCP protein, Fzd6, was asymmetrically localized to the anterior-ventral side of the apical cell membrane, downstream of CSF flow ( Figures 1B and 1C). A different core PCP protein, Vangl2 was specifically localized on the opposite side ( Figures 1B and 1C).
Immunofluorescence of cells stained with antibodies against tyrosinated a-tubulin showed that newly polymerized dynamic microtubules were located at the Fzd6 side of the cell cortex ( Figure 1D). Microtubule-like filaments were observed by electron microscopy to be connected to a protrusion from the BB that indicates cilia direction in ependymal cells, known as the basal foot (BF) ( Figure 1E). Tyrosinated tubulin recruits microtubule plus-end-tracking proteins (+TIPs) at the microtubule plus end (Peris et al., 2006). EB3 is a +TIP that co-localized with tyrosinated tubulin at the anterior-ventral side of the ependymal cell cortex ( Figure 1F). In contrast, CAMSAP2, a microtubule minus-end binding protein, was attached to the ciliary base ( Figure 1G). To determine the functional role of a microtubule connection from BB to the Fzd side of the cell cortex, tissue explants from the LV wall were treated with nocodazole, an inhibitor of microtubule polymerization. Microtubule dynamics were suppressed by nocodazole treatment and resulted in reduced tyrosinated tubulin with the BB positioned toward the anterior-ventral side of the cell cortex ( Figure 1H). Measured distance between the BB and the cell cortex was significantly larger (p = 0.0002) under nocodazole treatment (0.4929 G 0.02465, n = 54) compared with the control (0.3713 G 0.02013, n = 59) ( Figure 1I). These results suggest that BB positioning takes place through the extension of planar polarized microtubules from BB at its minus end toward the Fzd-side cell cortex at the plus end.

Daple Modulates Microtubule Dynamics at the Fzd Side of the Cell Cortex
Dvl is an Fzd-binding protein that acts as one of the core PCP proteins and is involved with cilia orientation in ependymal cells (Ohata et al., 2014: Yang andMlodzik, 2015). The three isoforms of Dvl (Dvl1-3) are localized differently in multi-ciliated cells (Vladar et al., 2012). Dvl2 was found to be localized at the BB in ependymal cells (Ohata et al., 2014), whereas Dvl1 co-localized with Daple at the anterior-ventral side of the apical cell cortex ( Figure 2A). Daple is a Dvl-associating scaffold protein that functions in non-canonical Wnt signaling and is required for BB positioning and orientation (Ishida-Takagishi et al., 2012;Takagishi et al., 2017). We have previously found that Daple co-localizes with Fzd6 at the anterior-ventral side of ependymal cells and is required for planar polarization of microtubules (Takagishi et al., 2017). In this study, we observed co-localization of Fzd6, Dvl1, and Daple with tyrosinated tubulin at the anteriorventral side of ependymal cells ( Figure 2B). Additionally, at the LV wall of both wild-type (Daple +/+ ) and Daple knockouts (Daple À/À ), tyrosinated tubulin was also localized to the cell cortex in ependymal cells with asymmetric accumulation of Fzd6 ( Figure 2C). Daple À/À ependymal cells significantly decreased (p < 0.0001) localization of tyrosinated tubulin at the Fzd6 side of the cell cortex (0.9785 G 0.04882, n = (C) Confocal microscopy images of the apical surface of Daple +/+ or Daple À/À ependymal cells on the LV wall stained with antibodies against Fzd6 (green), tyrosinated tubulin (red), and g-tubulin or b-catenin (white). Arrowheads indicate the accumulation of Fzd6 or tyrosinated tubulin at the cell cortex. Dashed lines indicate BB clusters. (D) Accumulation of tyrosinated tubulin at the Fzd6 side of the cell cortex was quantified for Daple +/+ and Daple À/À ependymal cells. The mean intensity of tyrosinated tubulin at the Fzd6 area of the cell cortex was normalized against the mean intensity of tyrosinated tubulin for the entire cell surface area. Normalized intensities are plotted as mean values G SEM for 30 ependymal cells, sampled from three mice in each group. (E) Kymographs of single microtubules from Daple +/+ or Daple À/À ependymal cells. The tips of EMTB-EGFP at the cell edge were monitored by fluorescence time-lapse microscopy imaging, see also Videos S1 and S2. (F) Quantification of the state of microtubule dynamics in Daple +/+ or Daple À/À ependymal cells. Shrinking, growing, and pausing (pause defined as > 0.1 mm/2 s) states of 30 microtubules were measured over 2.55 min for each group. Values are represented in Table S1.
iScience 23, 101213, June 26, 2020 iScience Article 30) relative to Daple +/+ (1.824 G 0.05945, n = 30) ( Figure 2D). This suggests that Daple is involved in the recruitment of the end of the microtubule where dynamic changes take place to the Fzd side of the cell cortex.
As Daple À/À ependymal cells had decreased tyrosinated tubulin (Takagishi et al., 2017), we proceeded to determine the effect of Daple deficiency on microtubule dynamics in ependymal cells. Polymerization dynamics at the end of individually labeled microtubules were monitored at the cell cortex in primary cultured ependymal cells expressing E-MAP-115 microtubules-binding domain (EMTB)-EGFP (Faire et al., 1999). In Daple +/+ ependymal cells, microtubules at the cell cortex showed continuous growth and shrinkage (Video S1). In contrast, microtubules in Daple À/À ependymal cells showed only minimal growth and shrinkage (Video S2). The changes at the end of a representative microtubule are plotted against time ( Figure 2E). The wild-type Daple +/+ microtubule frequently oscillated between growth and shrinkage, whereas Daple À/À microtubules exhibited minimal oscillation. Quantification of the changes in microtubule dynamics showed that Daple À/À had significant increases (p < 0.0001) in time spent in the pausing state and decreased (p < 0.0001) time spent in the shrinking or growing state ( Figure 2F and Table S1). These results suggest that Daple promotes dynamic microtubule changes, which explains the co-localization of Daple with tyrosinated tubulin at the Fzd side of the cell cortex. This further begs the question of how local microtubule dynamics regulate BB positioning.

Cytoplasmic Dynein Is Anchored to the Cortex in Ependymal Cells
In mitotic cells, cytoplasmic dynein anchored at the cell cortex captures the dynamic end of the microtubule and generates a pulling force on astral microtubules connected to the spindle pole (Laan et al., 2012;McNally, 2013). During ependymal cell maturation (P7-P15), BBs are strongly pulled toward the anterior side of the cell cortex, where Fzd has accumulated (Hirota et al., , 2016. We hypothesized that dynein's pulling force was also generated on planar polarized microtubules that were connected to BBs. Wholemount immunofluorescence imaging of the LV wall showed that dynein intermediated chain (IC) accumulates at the apical cell cortex, with tyrosinated tubulin on the anterior-ventral side of LV ( Figure 3A). Dynein IC was also observed at the BB cluster.
Daple has also been reported as an adaptor protein of cytoplasmic dynein (Redwine et al., 2017). We used co-immunoprecipitation of LV tissue lysate to determine if Daple interacts with dynein. Dynein IC, heavy chain (HC), and Dvl1 co-immunoprecipitated with Daple ( Figure 3B). Daple co-immunoprecipitated with dynein IC but Dvl1 did not ( Figure 3C).
We visualized the localization of dynein and Daple mutants in primary cultured ependymal cells to obtain more information about dynein's anchoring proteins at the cell cortex. The N-terminal (NT) region of Daple binds to dynein (Redwine et al., 2017). Daple interacts with a heterotrimeric G-protein Gai by binding to its Gai-binding and activating (GBA) motif, and Dvl by binding to its C-terminal PSD95/Dlg/ZO-1 (PDZ)-binding motif (PBM) ( Figure S1A; Aznar et al., 2015). Daple wild-type, DNT, and DGBA, but not DPBM, were localized at the cell cortex ( Figure S1B). GFP-dynein was localized at the cell cortex with Daple wild-type (76.5%) or DGBA (67.5%) but not with Daple DNT (7.1%) or DPBM (6.5%) (Figures S1B and S1C and Table  S2). These results suggest that PDZ binding of Daple is necessary for the anchoring of dynein to the cell cortex in ependymal cells. We observed the co-localization of dynein with Daple in the cortex of ependymal cells at the anterior-ventral side of the Daple +/+ LV wall ( Figure 3D). Dynein co-localization was abolished in the Daple À/À LV wall, with disrupted BB positioning and orientation (Takagishi et al., 2017). Taken together, these data indicated that Daple is required for anchoring of cytoplasmic dynein to the anterior-ventral side of the cell cortex.

Cytoplasmic Dynein Regulates BB Position and Orientation
To determine if cytoplasmic dynein is involved with BB positioning and orientation, tissue explants from the LV wall were treated with an inhibitor of cytoplasmic dynein, Dynarrestin (Hö ing et al., 2018). Dynarrestin treatment disrupted BB positioning and recruitment of tyrosinated tubulin to the cell cortex ( Figure 4A) and significantly increased (p < 0.0001) the distance between the BB cluster and the cell cortex (0.5381 G 0.01875, n = 58) relative to the DMSO control (0.3713 G 0.02013, n = 59) ( Figure 4B). Rotational BB orientation was visualized by immunofluorescence staining with antibodies against FGFR1 oncogene partner (FOP) and g-tubulin ( Figure 4C) and was defined as the angle from FOP to g-tubulin dots ( Figure 4D See also Figure S1 and Table S2.  Figure 4E). These data suggest that cytoplasmic dynein also regulates BB positioning and orientation in ependymal cells.

DISCUSSION
The role that PCP proteins play in microtubule polarization has long been a subject of speculation. It was widely assumed that downstream cytoplasmic molecules of Wnt/Fzd signaling regulate microtubule iScience Article remodeling; however, molecular mechanisms that regulate microtubule polarity remain to be elucidated. Here, we used ependymal ciliated cells in well-polarized LV wall tissue as a model and found that core PCP proteins Fzd/Dvl and Daple anchor dynein to the cell cortex and generate a pulling force on dynamic microtubules to exert force on BBs ( Figure 4F). As such, BB positioning and cilia orientation are regulated at the molecular level by PCP proteins.
Translational BB position is spatiotemporally altered in ependymal cells at the LV wall during development and aging (Hirota et al., 2016). We have shown that stable tyrosinated tubulin localizes to the Fzd side of the cell cortex in ependymal cells ( Figure 1D). Stable tyrosinated tubulin reflects microtubules at a state of dynamic equilibrium and includes tubulin dimer polymerization and depolymerization. Dynein at the cortex generates a pulling force on depolymerizing microtubules toward the cell cortex (Laan et al., 2012). On the contrary, microtubules undergoing growth can generate a pushing force (Vleugel et al., 2016). Both shrinking and growing of microtubules were suppressed in Daple-deficient ependymal cells ( Figure 2F), which led to the disruption of translational BB position (Takagishi et al., 2017). These findings provide evidence for a model where continuous microtubule dynamics fine-tune the BB position by modulating the distance between the BB and the cell cortex.
BB position plays an important role in the rotational orientation of cilia. Translational positioning and rotational orientation of BB are established at around the same developmental stage, during P9 . We have shown that treatment with a dynein inhibitor suppressed not only BB positioning but also its orientation ( Figure 4). During translational positioning, dynein anchored on the Fzd side can generate a pulling force on the depolymerizing microtubules connected to the BF. The pulling force enables the orientation of the BF toward the Fzd side and leads to the rotational orientation of BBs.
Having found that Daple modulated microtubule dynamics at the Fzd side of the cell cortex ( Figures 2C-2F), we proceeded to define the molecular mechanisms involved in this process. Dynein that is anchored to the cell cortex is able to capture the plus end of microtubules and control microtubule dynamics (Laan et al., 2012). As we observed that dynein inhibition decreased tyrosinated tubulin in ependymal cells (Figure 4A), we hypothesized that Daple might act through anchored dynein to promote dynamic cycling of microtubules at the cell cortex in ependymal cells.
It is known that core PCP proteins are also involved in the orientation of hair bundles on auditory sensory cells (Ezan and Montcouquiol, 2013). It has previously been reported that Daple regulates cilia orientation not only in multi-ciliated cells at the LV wall or in the trachea but also in hair cells of the auditory system (Takagishi et al., 2017;Siletti et al., 2017). In auditory sensory cells, Daple is asymmetrically localized at the cell cortex, on the same side as Fzd/Dvl. Interestingly, despite normal localization of Dvl and Gai, Daple knockout mice showed disrupted kinocilium positioning in hair cells (Siletti et al., 2017). As microtubules and dynein regulate BB positioning of the kinocilium (Lu and Sipe, 2016), it is possible that Daple may anchor dynein to pull the BB of the kinocilium toward the Fzd/Dvl side of the cell cortex. This hypothesis is supported by evidence from a yeast two-hybrid assay, which showed a cofactor of dynein, dynactin, interacts with Daple in auditory sensory cells (Siletti et al., 2017). Taken together, these studies suggest that Daple may play an important role in various tissues in the PCP protein-mediated regulation of BB positioning through cortical dynein.
Critically, the data here demonstrate that the C-terminal PDZ-binding motif of Daple is essential for the anchoring of Daple and dynein to the cortex ( Figure S1). A nonsense mutation E1949GfsX26 in DAPLE is found in patients with hydrocephalus and causes termination at 1973, which results in the loss of the PDZ-binding motif (Drielsma et al., 2012). Daple-deficient mice showed loss of dynein at the cell cortex (Figure 3E), and lack of coordinated cilia orientation and movement on the LV wall, resulting in stagnant CSF flow and hydrocephalus (Takagishi et al., 2017). This mouse model study not only improves our understanding of mechanisms underlying cilia orientation but may also provide insight into the etiology of human hydrocephalus.

Limitation of the Study
In this study, we cultured ependymal cells to observe microtubule dynamics and the cortical localization of dynein with Daple mutants (Figures 2E, 2F, and S1). However, these cultured ependymal cells could not be perfectly polarized under no-flow conditions ( Table S2.  Figure S1 and Figure 3.

Animal experiments
C57Bl/6J mice were used for whole-mount staining, ependymal cell culture, and LV explants cultures. ES cell clone (DEPD00564-1-E06) for Daple knockout mice purchased from the trans-NIH KOMP Repository (University of California Davis) (Skarnes et al, 2011). All mice were maintained with 12/12 h light/dark cycle and no more than 5 mice per cage. Both male and female mice were used for the experiments, and sex differences were not observed. All animal experiments were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine (approval no. 30364) and conducted in accordance with guidelines.