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Seminars in Cell & Developmental Biology xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Seminars in Cell & Developmental Biology
journal homepage:
Role of the crumbs proteins in ciliogenesis, cell migration and actin
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
Polarity complexes
Cell polarity
Cell migration
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.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Crumbs complex and ciliogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Crumbs complex and cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Chemical cues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
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: (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
1084-9521/© 2017 Published by Elsevier Ltd.
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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
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)
Please cite this article in press as: E. Bazellières, et al., Role of the crumbs proteins in ciliogenesis, cell migration and actin organization,
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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.
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 ».
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