Role of the crumbs proteins in ciliogenesis, cell migration and actin organization

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Review

Role of the crumbs proteins in ciliogenesis, cell migration and actin organization Elsa Bazellières, Veronika Aksenova, Magali Barthélémy-Requin, Dominique Massey-Harroche, André Le Bivic ∗ Aix-Marseille University, CNRS, IBDM, Case 907, 13288 Marseille, Cedex 09, France

a r t i c l e

i n f o

Article history: Received 11 July 2017 Received in revised form 9 October 2017 Accepted 18 October 2017 Available online xxx Keywords: Polarity complexes Cell polarity Cell migration Cytoskeleton

a b s t r a c t Epithelial cell organization relies on a set of proteins that interact in an intricate way and which are called polarity complexes. These complexes are involved in the determination of the apico-basal axis and in the positioning and stability of the cell–cell junctions called adherens junctions at the apico-lateral border in invertebrates. Among the polarity complexes, two are present at the apical side of epithelial cells. These are the Par complex including aPKC, PAR3 and PAR6 and the Crumbs complex including, CRUMBS, PALS1 and PATJ/MUPP1. These two complexes interact directly and in addition to their already well described functions, they play a role in other cellular processes such as ciliogenesis and polarized cell migration. In this review, we will focus on these aspects that involve the apical Crumbs polarity complex and its relation with the cortical actin cytoskeleton which might provide a more comprehensive hypothesis to explain the many facets of Crumbs cell and tissue properties. © 2017 Published by Elsevier Ltd.

Contents 1. 2. 3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Crumbs complex and ciliogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Crumbs complex and cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Chemical cues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Physical cues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Crumbs complex and the actin cytoskeleton: a unifying theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Future perspectives and concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Abbreviations: AJ, adherens junction; TJ, tight junction; aPKC, atypical protein kinase C; CRB, crumbs; DLG, discs large; ECM, extracellular matrix; FERM, 4.1 ezrin radixin moesin; LGL, lethal giant larvae; MAGUK, membrane-associated guanylate kinase; MUPP1, multi PDZ domain protein; Ome, oko meduzy; PALS, protein associated with Lin seven; PAR, partition defective; PATJ, PALS1-associated tight junction protein; PDZ, PSD-95 discs large ZO-1; SCRIB, scribble; Sdt, stardust; SH3, Src homology domain 3. ∗ corresponding author. E-mail address: [email protected] (A. Le Bivic).

Cell polarity is a general feature of living cells, from bacteria to eukaryotes. Overall cell polarity is linked to the necessity to move, to divide or to function directionally. Multicellularity has however introduced an additional level of organization as cell polarity and movements have to be coordinated at the level of the tissue [1]. This is particularly true for metazoans since morphogenetic events such as gastrulation that are essential for morphogenesis, involve coordinated cell movements and coupling of cell forces while keeping the homeostasis of the developing organism [2]. To achieve these complex morphogenetic events, metazoans have developed a new tissue organization with epithelial layers that are made of a single

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sheet of polarized adherent cells. In epithelia, each cell has a polarity which is integrated in a higher order of polarized organization of the tissue. Several years of research have led to define cell polarity in epithelial cells within two axes: The Planar Cell Polarity (PCP) and the Apico-Basal Polarity (ABP). PCP coordinates in the plane of the epithelium the asymmetric distribution of several cell features, such as actomyosin cytoskeleton organization or cilia positioning, necessary for movement, feeding or sensing (for review see [3]). This polarity relies on a set of proteins called the PCP core complex made of several transmembrane proteins (Flamingo, van Gogh . . .) and adapters such as Prickle or Disheveled (for review see [4]). The other polarity system is the one that defines the ABP within epithelial cells. ABP is based on the formation of a free cell surface in contact with the external medium (the apical side), cell–cell contacts in the lateral domain and a basal side that lies most often on a basement membrane, opposite the apical side. The apical side is separated from the lateral domain by a set of specialized cell–cell junctions, which preserve the organism homeostasis (for review see [5]). The integrity of the cell layers, in vertebrates, is mediated by the physical coupling of the cells through different sets of junctions, namely tight junctions, adherens junctions, and desmosomes [6]. Apical and basolateral membranes are characterized by the presence of protein and lipid markers such as channels, transporters or enzymes linked to the function of these membranes. While these proteins or lipids are usually strongly associated to a specific polarized domain most of them do not play an instrumental role in the establishment or maintenance of a polarized epithelium. Only a set of few proteins or lipids has been identified to play a role in establishing and/or maintaining epithelial ABP and organization [7,8]. The first set of genes involved was discovered using the Caenorabditis elegans model and genetic screens that identified Par proteins (for partitioning defective) including the Par3/Par6/aPKC (atypical protein kinase C) apical complex and the lateral Par1/Par4 complex [9,10]. For the polarity to be established, the Par6/Par3/aPKC and Par1 mutually exclude each other through antagonistic phosphorylation. This will actively drive the segregation of the Par polarity protein into their respective apical and basolateral domains [11]. Once the polarity established, these complexes regulate the actin cytoskeleton and the endocytosis providing thus a mean to maintain distinct apico-basal cortical and membrane subdomains [12]. Another complex involved in ABP is the lateral Scribble complex identified in flies [13] and made of Scribble, Discs large (Dlg) and Lethal giant larvae (Lgl) (for review see [14]). This complex is involved in vesicular trafficking and cell proliferation (for review see [15]). In addition to these cortical or cytoplasmic complexes, a membrane anchored complex is formed by Crumbs, an apical transmembrane protein [16], stardust (PALS1,Protein Associated to Lin Seven, in mammals), an adaptor of the MAGUK (Membrane Associated GUanylate Kinase) family [17,18] and Patj (PALS1-Associated TJ protein), a protein containing multi PDZ (PSD-95, Discs large, ZO-1) domains [19,20]. This was the first core Crumbs complex identified and later it was shown in vertebrate that CRUMBS itself can bind directly to PAR6 [21] and that in Drosophila aPKC phosphorylates Crumbs cytoplasmic tail [22] suggesting that they might form another complex together. Moreover, it was shown that Stardust/PALS1, PATJ and Par6 also interact together [23,24], blurring the distinction between two distinct Crumbs complexes. The core Crumbs complex is involved in the regulation of the cortical actin cytoskeleton [25], the stabilization of AJs [26], vesicular trafficking [27] and cell proliferation [28,29]. For a more detailed description and functional analysis of the Crumbs complexes we suggest several recent reviews [27,30]. In this review, we will focus on the role of the Crumbs complex in less explored functions or in fast moving aspects of its cell biology.

2. Crumbs complex and ciliogenesis Cilia are extensions of the apical surface of most quiescent and differentiated cells (for review [31]). In most cases, primary ciliogenesis begins by the gathering of small vesicles originated from the Golgi apparatus that reach the activated mother centriole using a polarized endosomal trafficking [32]. Fusion of these vesicles produces a membranous cap called the ciliary vesicle at the distal tip of centriole. From this distal tip, microtubules grow in a polarized manner under the cap that jointly increases due to the addition of membrane. This nascent axoneme is therefore inserted in a double membrane which fuses with the apical plasma membrane during the emergence of the cilium. In epithelial cells, however, cilia grow directly by extension of the apical membrane around the axoneme (for review see [33]). Like all organelles, the cilium is maintained by polarized vesicular traffic within the cell and along the axonemal microtubule network, with the specific molecular intraflagellar transport machinery [34]. Crumbs proteins and the polarity Par complex that specify apical identity have been involved in epithelial ciliogenesis (Fig. 1). The first Crumbs involved in ciliogenesis was CRB3 and in mammals, the CRB3 gene codes by alternative splicing for two isoforms: CRB3A with the canonical COOH-terminal ERLI motif and CRB3B with a COOH-terminal CLPI motif. These two isoforms are localized in cilia of MDCK cells (Madin Darby Canine Kidney cells) and are involved in its formation [35,36]. This is also the case for the polarity Par complex (PAR6, PAR3 and aPKC) which co-localizes to the primary cilium in the same cells and it has been proposed that CRB3A and the Par complex interact in the cilium [36]. Previously, we have identified an interaction between CRB3A and PAR6␣ via the PDZ binding domain (ERLI) of CRB3A and the PDZ domain of PAR6␣ [21] thus providing a direct link between these two complexes involved in ciliogenesis. While CRB3A is involved in the initiation of ciliogenesis, PAR3 (linked to KIF3A/kinesin2/microtubules) seems to participate to the anterograde vesicular transport for the elongation of primary cilia [37] suggesting that CRB3A is required for the delivery of the Par complex to the cilium and acts upstream of it. It is interesting to mention that PAR6γ is also present at the centrosome suggesting that it could act earlier in ciliogenesis than proposed by organizing the pericentriolar domain [38]. CRB3B (also called CRB3-CLPI) does not interact with the Par complex but its targeting to the cilium is mediated by importin ␤-1, a nuclear import protein, that is essential for cytokinesis but also for ciliogenesis [35]. Despite the fact that CRB3A or B have been involved in ciliogenesis a decade ago the molecular mechanisms at work remain unclear. In addition to be involved in primary ciliogenesis, CRB3 is necessary for the multiciliated airway cell differentiation [39] but a direct role of the Crumbs proteins in multiciliogenesis has not been demonstrated yet. It must be however noted that CRB2B (one of the CRB2 proteins in zebrafish) accumulates at the basis of the ciliary tuft in pronephric cells and that CRB2B knock-down induced a strong reduction in cilium length indicating that it plays a role in the formation or maintenance of cilia in multiciliated cells [40]. Some cells possess cilia with specialized sensory functions and it is the case for retina photoreceptors which bear inner and outer segments on their apical side (Fig. 1). This specialized cilium is in constant renewal throughout life while photoreceptors are not renewable. Several studies from flies to man have shown that Crumbs proteins are essential for proper photoreceptor morphogenesis and survival [41–43] and are involved in the building of this specialized structure both in the zebrafish and in mammals. In zebrafish, CRB2A is expressed in the inner segments of all types of photoreceptors and in the apical domain of Müller cells whereas CRB2B (also called Oko meduzy or Ome) [40] is mainly expressed in the inner segments of green, red and blue cones [44]. CRB2A is involved in the regulation of the inner segment size as over-

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Fig. 1. Crumbs complex and ciliogenesis. The localization of the different CRUMBS and their binding partners are represented in the different types of cilia. On the left a photoreceptor with a connecting cilium is shown, in the middle a cell with a primary cilium and on the right a multiciliated cell. In all cases, CRUMBS proteins are localized at the level of the junctions, Sub-Apical Region (SAR) of the photoreceptor and tight junctions of the other cells. CRUMBS proteins function to organize the apical membrane and underlying cytoskeleton (green) and have also been shown to be involved in polarized vesicular trafficking in the photoreceptor (from the SAR to the connecting cilium) and in the primary cilia.

expression of full-length protein induced an increase in its size [45]. In human and mouse photoreceptors, CRB1 and 2 are localized to the inner segments in addition to the cell–cell junctions (for review see [46]). It is of particular interest that CRB2 is accumulated in vesicles in the striated ciliary rootlets at the tip of the inner segments suggesting that CRB2 could play a role in transporting material to the connecting cilium [42]. CRB3A is also found in the vicinity of the connecting cilium of human photoreceptors [47,48] indicating that it might also have a conserved role between the primary cilium and the connecting cilium. Both CRB1 and CRB2 when mutated induced retina pathologies with photoreceptor degeneration [49] but the mechanism behind this degeneration is not known. One hypothesis could be that the lack of either CRB1 or 2 might impair transport of essential components towards the outer segment through the connecting cilium. In Drosophila melanogaster, transport of rhodopsin is based on Myosin V that is in turn stabilized by Crumbs [50]. It must be noted that in mouse photoreceptor inner segments Myosin V is also detected but its function in photoreceptors has not been addressed [51]. Thus, more work is necessary to understand the molecular role of Crumbs proteins in ciliogenesis and photoreceptor morphogenesis and survival. 3. Crumbs complex and cell migration Cell migration is an important process that occurs in several events during either development, adulthood or pathological conditions. Cells can migrate as single units or collectively. Collective cell migration is an efficient process as a cluster of cells move in the same direction with a similar speed, compare to isolated cells that undergo a less persistent migration with frequent changes in their direction. In all these situations, polarity proteins are essential, as they will dictate how the cells will migrate. The level of expression together with the localization of the Par and Crumbs complexes strongly correlate with epithelial cell behavior, and with the balance between a static differentiated epithelium and a loosely connected/collectively migrating cells. The expression and local-

ization of the polarity protein PATJ, PALS1 (both members of the Crumbs complex) and PAR6, PAR3, aPKC confer the migration property of the cells as their accumulation at the leading edge will result in a polarized/directed and persistent migration whereas their mislocalization will give rise to a random migration (for review see [52–55]). Even though several studies have demonstrated an implication for PATJ, PALS1 in cell migration, the role of CRUMBS during both single and collective cell migration still needs to be clearly demonstrated. CRUMBS as a transmembrane protein that can recruit the cytoplasmic proteins PAR6 and PALS1 at the cell membrane can promote the formation of different polarity complexes. Thus, CRUMBS could participate in the recruitment of these complexes at the leading edge of migrating cells. This localization is essential for the initial breaking of symmetry that leads to cell polarization. The temporal regulation also needs to be elucidated but it has been proposed that PATJ can recruit PAR3 and aPKC at the wound edge [54], where it can be activated by Cdc42, thereby initiating downstream events such as stabilization of microtubules or integrins [56–58]. We have recently identified a new interactor of PAR6␣, HOOK2, a microtubule binding protein [59]. In this study, we have unveiled a new function of HOOK2 in maintaining PAR6␣ at the centrosome level, resulting in an efficient and polarized migration of the epithelial sheet. From all these different studies, it seems to be important to look at the role of the polarity complexes not only at the level of the leading edge, but also elsewhere in the cells, as they are involved in the stabilization of the polarized organization of the cells during migration. During the so-called Epithelial to Mesenchymal Transition (EMT), it has been described in several models, in vivo and in vitro, that the polarity complexes Crumbs and Par are perturbed within their localization or expression. In these contexts, the polarity complexes play different roles. Historically, it has been admitted that CRUMBS could act as a tumor suppressor, as its expression is frequently lost in advanced tumors [60–64]. Recent works show, however, that the loss of different isoforms of CRUMBS, namely CRB3 and CRB2 can induce or prevent the EMT to occur, respec-

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tively. For instance, loss of CRB3 expression in non-tumorigenic human mammary epithelial cells increases cell invasion, activates the transcription factor Snail and promotes cell scattering [64]. In contrast, during mouse gastrulation the loss of CRB2 expression prevents the disassembly of AJs leading to defect in cell ingression [65]. In both cases, the consequence is an impairment of the dynamical remodeling of the cell–cell adhesions, which could lead to a weakening of cell–cell adhesion in the CRB3 depletion or to a strengthening of cell–cell adhesion in the CRB2 depletion, resulting in an existing but perturbed cell migration. In contrast to the Crumbs complex, in cancer cells, proteins of the Par complex are overexpressed or mislocalized and then could potentially act as oncoproteins [66,67]. Interestingly, the overexpression of any member of the Par complex also leads to an impairment of TJs integrity and apico-basal polarity, which could mechanically also result in the weakening of cell–cell adhesion. Recently, this idea has been challenged by a bioinformatic study, where it has been demonstrated that CRB1, CRB2 or PAR6 gene expressions are downregulated in several cancers, whereas, in the same cancer types, CRB3, PAR6˛ and PAR6ˇ are upregulated [68]. By expanding the analysis to all the members of the polarity complexes, it was concluded that polarity complexes play an important role in tumor progression, although the specific effects on depletion or upregulation are cancer type dependent. All the studies done so far have clearly established a link between the behavior of migrating cells and the polarity complexes Crumbs and Par. However, the key events and factors that trigger the correct level expression or localization of the Crumbs and Par complexes are still unclear. So far, it has been described that during migration, by responding to different cues such as chemical (soluble factors, composition of the matrix) or physical (pulling forces, release in tension), epithelial cells can move persistently in a given direction correlating with the accumulation of the polarity complexes Crumbs and Par at the leading edge (for review see [52,53]). The potential impact of chemical or physical cues on polarity protein expression or localization is discussed in the next sections. 3.1. Chemical cues During cancer progression, it has been shown that the growth factor TGF␤ can dictate and enhance the occurrence of EMT through its effect on polarity proteins, such as phosphorylation of PAR6 [69] or the downregulation of PAR3 and CRB3 [70,71]. Indeed, TGF␤ associates and phosphorylates PAR6␤, resulting in TJs dissolution [69] and in the formation of the PAR6␤/aPKC complex at the lea ding edge [72]. In the later study, it was further shown the importance of such a localization for the formation of the PAR6␤/aPKC complex that connects to the microtubule system and directs cell migration. The effect of TGF␤ will thus impact the tension at the cell–cell interface by weakening the adhesions and by stabilizing microtubules. This will allow the occurrence of pulling force that reorient the microtubule network resulting in a persistent migration [73],[74]. The matrix composition is also important. Deregulation of the extracellular matrix (ECM) environment can disrupt ABP and promote collective cell migration. This occurs via changes in the expression of matrix metalloproteinases, integrins, and ECM proteins (review in [75]) that correlate with changes in expression and/or localization of polarity proteins. When epithelial cells acquire a migrating phenotype, the Par complex is re-localized at the anterior/basal domain of the epithelial migrating cells, whereas CRUMBS expression is decreased but how this is triggered by the matrix is still unclear. Some evidences clearly point out a link between matrix composition and polarity complexes. As an exemple, in pancreatic carcinoma cells, collagen I and integrin expression are increased leading to AJs disruption and nuclear translocation of ␤-catenin [76]. This nuclear translocation of ␤-catenin by activating

the transcription factor Snail has been shown to impact and remove the junctional localization of PAR3 and aPKC without affecting their expression. It was further showed that Snail activation repress CRB3 expression whereas PALS1 and PATJ expression were only reduced [70,77]. In that context, it is tempting to speculate that the remaining PAR3, aPKC, PALS1 and PATJ could be relocalized to the leading edge allowing efficient cell migration. 3.2. Physical cues Interestingly, increasing evidences have shown that the microenvironment can influence tissue polarity and promote collective cell migration. Notably, the change in ECM composition is known to influence the rigidity of the matrix. During cancer progression, an increase in matrix rigidity has been extensively shown in several models [78]. This increase in rigidity has been demonstrated to disrupt tissue polarity and promote collective cell invasion, a process called durotaxis [78–80]. During durotaxis, cells migrate persistently toward the stiffer matrix, and acquired a spread and polarized shape, that is associated with a high Rac, RhoA, ROCK and Cdc42 activity [81–83]. Even if the link with the polarity complexes is not clearly established, PAR6 is a known interactor of Cdc42 and CRB3. It is tempting to speculate that the rigidity will impact the localization of these complexes toward the leading edge. Furthermore, an increase in rigidity has been described to modulate gene expression and cytoskeletal architecture favoring the EMT [78]. This EMT strongly correlated with changes in expression or loss of functional activity of the cell polarity complexes Crumbs and PAR6, reinforcing the functional link between rigidity and polarity complexes [60–62,68]. During migration, the formation of a free edge can also be thought as a release of lateral tension together with the weakening of the cell–cell junction [84,85]. This process has been described to happen in vivo when the gut suffers mild injuries [86]. During this process, epithelial cells from the intestine acquire a migrating phenotype, and proteins, such as villin, relocalize from the apical membrane toward the leading edge [86]. In this context, it would useful to understand how the Crumbs and Par complexes behave and if the release in tension is sufficient to drive a change in localization of the polarity proteins. Interestingly, Merlin has been described to be sensitive to tension. A change in tension at the cell–cell interface, when the migrating cells are pulling on the cell behind, has been shown to remove Merlin from the cell–cell junction, allowing the generation of a Rac gradient needed for the formation of the lamellipodia [87]. However, the link with the polarity complex CRUMBS/PATJ/PALS1 is not clear in this study, even if some other studies suggest an indirect link between CRUMBS and Merlin through either PAR3 [88] or Expanded [89,90]. Nowadays, the interplay between the nanotechnologies (tuning of the matrix), the physics (measuring and applying forces) and the biology give the opportunity to tackle all the remaining questions, and to go further in the understanding of the implication of polarity protein during cell migration. 4. Crumbs complex and the actin cytoskeleton: a unifying theory So far, the function of Crumbs complex has been compartmentalized to different processes, namely the formation and maintenance of epithelial junctions, cell proliferation, ciliogenesis, and migration (for review see [27]). However, all these processes require changes in cell shape (Fig. 2) which are intrinsically linked to a specific organization and turnover of the actin cytoskeleton. When epithelial tissue polarity switches from an apico-basal (nonmigratory state, formation and maintenance of junctions and cilia)

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Fig. 2. Crumbs complex and actin organization during apico basal differentiation and cell migration. The localization of the Crumbs complex is affected by external cues such as matrix stiffness or TGF␤ signaling. On soft substrate, CRUMBS is localized apically where it triggers different signaling pathways, such as activation of Cdc42 that will allow the dynamics of the TJs and will impact the activity of the actin binding partner that bind to CRUMBS. All these interactions will allow the reorganization of the actin cytoskeleton and its contraction thanks to the localized activity of Rho, generating apical forces needed for the cell to acquire their columnar shape. Another protein platform formed by CRUMBS is the one constituted of phosphorylated Merlin and phosphorylated Yap. In this configuration, these proteins are inactive allowing cell differentiation. On stiff substrate, the Crumbs complex is localized at the leading edge together with Par complex, Rac and Cdc42, allowing the formation of the lamellipodia. The Par complex is also localized at the centrosome where it interacts with Hook2, a microtubule binding protein. The generation of basal and junctional forces impacts the localization of YAP and Merlin, which are translocated to the nucleus and remove from the front edge of the pulled cells respectively. These mechano-translocation and mechano-delocalization result in the activation of YAP and Merlin that will result in the expression of gene needed for the migration and in the activation of Rac at the leading edge. How CRUMBS proteins behave during migration is still unclear and we propose two scenarios, one where CRUMBS is also relocalized to the leading edge and one where the forces break the bond between CRUMBS/Merlin and CRUMBS/YAP.

to an anterior-posterior (migratory state) polarity, epithelial cells need to change their architecture. By doing so, the cells adapt to potential changes in the surrounding environment as observed during cancer progression or differentiation [78,91]. Many years of research, and in particular the last decades have revealed an increase number of proteins that link CRB2 and CRB3 to several actin binding proteins. The proteins involved can interact with the FERM or the PDZ-binding domains of CRUMBS directly or indirectly through different partners. The final result of all these interactions can lead to a protein platform anchored to the cell membrane by CRUMBS. Here we will review the interactions between CRUMBS proteins and actin-binding proteins. Several years ago, our group has identified in flies an interaction between Crumbs and Moesin/␤-heavy-Spectrin demonstrating for the first time a crucial role for Crumbs in the stabilization of actin

cytoskeleton [25]. Since then, this interaction has been shown to regulate the apical constriction and cellular movement allowing the formation of the tracheal tube, or dorsal closure [92–94]. In mammalian systems, CRB2 or CRB3 can interact with the actin cytoskeleton, through interactors such as E-cadherin [95], Moesin [25], Ezrin [96], Arp2/3, Eps8 [97] or EHM2 [98] but also through a co-regulation between these polarity complexes and the small GTPases, Rho, Rac and Cdc42. Recent studies have demonstrated that cells depleted for CRB3 possess truncated actin microfilaments with a decrease expression levels of formin1 [97], and leads to membrane blebbing that is associated to a detachement of the actin cortex from the membrane [99,100]. Based on all these studies, it is clear that at least CRB3 and CRB2 can regulate the actin dynamics in different manners. It can either allow the formation of branched actin by recruiting Arp2/3 and activates actin nucleation through

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Rac1 and Cdc42 regulation or the formation of actin bundles by recruiting Eps8 and promotes actomyosin contraction by regulating Rho activation through EHM2. Even though the spatiotemporal regulation of all these proteins with CRUMBS is still unclear few studies have adressed this point. So far it has been demonstrated that Cdc42 is important for the correct localization of Crumbs [101] and for its interaction with Par6 [102], allowing the establishment of the apical domain. The correct localization of activated Rac and Cdc42 is also important for the formation and stabilization of AJs and TJs [103–105]. During migration, these local activations induce cytoskeletal rearrangements and rapid actin polymerization that lead to the formation of membrane protrusions and promote engagement of integrins with the extracellular matrix [106]. aPKC, a component of the PAR6 complex, is a downstream effector of activated Cdc42, and has been shown to be localized at the cell front by PATJ, a component of the Crumbs polarity complex [54]. When localized at the cell front, aPKC will promote actin assembly by reinforcing different pathway such as the Tiam1-Rac1 signaling pathway [107]. In contrast to Rac and Cdc42, Rho is localized at the rear of the cell where it allows actomyosin contraction, that helps the translocation of the cell body during cell migration. During migration, CRB2 has also been shown to play an important role in the localization of the contractile actomyosin network, allowing the extrusion of cells during mesodermal invagination [65]. Taken together, all these studies strongly suggest that polarity proteins may control cell shape and dynamics by significantly contributing to the localization and activation of different small GTPases both at the apical and front ends of cells (e.g. Cdc42, Rac1) as well as at their basal and rear ends (e.g. Rho). Interestingly, these different small GTPases are involved in the formation and stability of specific actin filamentous structures, such as meshlike actin and actin bundles networks. These actin networks will either produce pushing or pulling forces allowing epithelial cells to tune their shape. In cuboidal and columnar epithelia, actin meshlike networks have been shown to be essential to preserve and maintain the stability of AJs, and TJs through the regulation of endocytosis [108]. Furthermore, the apical localization of actin bundles has been proposed to be responsible for the contraction of the apical domain leading to columnar and tall cells [109,110]. In flat and migrating cells, the mesh-like network allows the formation of lamellipodia at the cell front for efficient cell migration. There, actin bundles are localized at focal adhesions at the cellsubstrate interface [106,111] or at AJs at the cell–cell interface [112] where they operate to reinforce adhesion sites [113,114]. Interestingly, actin dynamics result in the generation of forces that are intrinsically linked to mechanotransduction signaling, leading to important switches in cell behavior [114,115]. When cells generate high forces, the mechanosensitive proteins YAP and Merlin lose their apical and junctional localization allowing the cells to switch from being differentiated/ciliated toward a more migrating phenotype [87,114,116,117]. YAP is phosphorylated upon the activation of the Hippo pathway, resulting in the cytoplamic localization of YAP. In Drosophila, Crumbs regulates the Hippo pathway through its interaction with Expanded [90–118]. Recently, studies done in mammalian system have revealed that CRB3 interacts with phosphorylated YAP and Kibra [39] or indirectly with Merlin [88–90], allowing the differentiation of multiciliated cells and the formation of normal 3D acini in MCF10A [119]. The loss of CRB3 has been associated with defects in cell differentiation and the formation of acini with multiple lumen, the degradation of Kibra by the proteasome and the nuclear localization of YAP. This localization of YAP induces the transcription of several factors involved in cell migration. YAP is a well-known mechanosensitive protein that is affected by cell–cell adhesion, and cell-substrate forces but also substrate rigidity. If cells are plated on top of a stiff substrate,

the forces are increased at the cell-substrate interphase and thus transmitted to cell–cell adhesions. In this high force condition, it is tempting to speculate that the CRB3/YAP and the CRB3/Merlin bonds are also mechanosensitive and could be released upon an increase in forces, resulting in YAP translocation to the nucleus and Merlin accumulation in the cytoplasm. From all these recent studies, it is clear that CRUMBS is not only involved in the establishment and maintenance of ABP, but has a much broader function. The different CRUMBS isoforms emerge as essential players in the dynamics of actin remodeling by interacting with many actin binding partners. Due to the fact that the different partners bind to the same cytoplasmic domain of CRUMBS, spatiotemporal regulation of these interactions must occur and much remains to be learned about how these multifaceted interactions direct tissue homeostasis and morphogenesis. 5. Future perspectives and concluding remarks In this review, we have focused on less characterized functions of the CRUMBS family of proteins, showing that they might have a broader and more general function than expected. More dynamical studies are still needed to fully understand how polarity complexes work in an orchestrated, organized and finely regulated manner. Studies done so far in vertebrates are limited in terms of spatiotemporal regulation of the interaction between CRUMBS and its multiple partners, making the picture complex and yet incomplete. In order to fully understand the function of the different Crumbs polarity complexes, the visualization and characterization of their spatio-temporal interactions are mandatory. Using the CRISPRCAS9 technology combined with optogenetic tools to spatially and temporally control these interactions will help to finely described how and when the different partners interact. Furthermore, nanotechnologies and biophysical tools will allow to pinpoint the global mechanical impact of the Crumbs complex on cellular forces and how the external constraints affect the regulation of the Crumbs complex together with its specific interactome. In addition to these basic cellular functions there are now evidences that all CRUMBS proteins are important players in some human pathologies but so far very little information has been provided on the mechanisms involved given the complexity of working directly on tissue organization and physiology in human. The next challenge will be to use human derived mini-organs expressing some mutated forms of CRUMBS genes. Acknowledments We thank Christopher Toret for critical reading of this manuscript. The Le Bivic group is an “Equipe labellisée 2008 de La Ligue Nationale contre le Cancer” and is supported by the labex INFORM (grant ANR-11-LABX-0054), the ANR grant Ghearact (14CE13-0013), CNRS and Aix-Marseille University. EB was supported by « La Ligue Nationale contre le Cancer ». References [1] F. Julicher, S. Eaton, Emergence of tissue shape changes from collective cell behaviours, Semin. Cell Dev. Biol. 67 (2017) 103–112. [2] L.A. Davidson, No strings attached: new insights into epithelial morphogenesis, BMC Biol. 10 (2012) 105. [3] R.S. Gray, I. Roszko, L. Solnica-Krezel, Planar cell polarity: coordinating morphogenetic cell behaviors with embryonic polarity, Dev. Cell 21 (2011) 120–133. [4] J.R. Seifert, M. Mlodzik, Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility, Nat. Rev. Genet. 8 (2007) 126–138. [5] E. Rodriguez-Boulan, W.J. Nelson, Morphogenesis of the polarized epithelial cell phenotype, Science 245 (1989) 718–725. [6] S. Jonusaite, A. Donini, S.P. Kelly, Occluding junctions of invertebrate epithelia, J. Comp. Physiol. B 186 (2016) 17–43.

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