Protein Crumbs Research Articles

Diverse functions and pathogenetic role of Crumbs in retinopathy.

The Crumbs protein (CRB) family plays a crucial role in maintaining the apical-basal polarity and integrity of embryonic epithelia. The family comprises different isoforms in different animals and possesses diverse structural, localization, and functional characteristics. Mutations in the human CRB1 or CRB2 gene may lead to a broad spectrum of retinal dystrophies. Various CRB-associated experimental models have recently provided mechanistic insights into human CRB-associated retinopathies. The knowledge obtained from these models corroborates the importance of CRB in retinal development and maintenance. Therefore, complete elucidation of these models can provide excellent therapeutic prospects for human CRB-associated retinopathies. In this review, we summarize the current animal models and human-derived models of different CRB family members and describe the main characteristics of their retinal phenotypes.

Crb3 is required to organize the apical domain of multiciliated cells.

Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane.

Fructose-bisphosphatase1 (FBP1) alleviates experimental osteoarthritis by regulating Protein crumbs homolog 3 (CRB3)

PurposeTo identify the role of gluconeogenesis in chondrocytes in osteoarthritis (OA).Materials and methodsCartilage samples were collected from OA patients and C57 mice and were stained with Safranin O-Fast Green to determine the severity of OA. Periodic acid Schiff staining was used to characterize the contents of polysaccharides and SA-βGal staining was used to characterize the aging of chondrocytes. Immunohistochemistry and western blotting were used to detect fructose-bisphosphatase1 (FBP1), SOX9, MMP13, P21, and P16 in cartilage or chondrocyte. The mRNA levels of fbp1, mmp13, sox9, colX, and acan were analyzed by qPCR to evaluate the role of FBP1 in chondrocytes.ResultsThe level of polysaccharides in cartilage was reduced in OA and the expression of FBP1 was also reduced. We treated the chondrocytes with IL-1β to cause OA in vitro, and then made chondrocytes overexpress FBP1 with plasma. It shows that FBP1 alleviated the degeneration and senescence of chondrocytes in vitro and that it also showed the same effects in vivo experiments. To further understand the mechanism of FBP1, we screened the downstream protein of FBP1 and found that CRB3 was significantly downregulated. And we confirmed that CRB3 suppressed the degeneration and delayed senescence of chondrocytes.ConclusionsFBP1 promoted the polysaccharide synthesis in cartilage and alleviated the degeneration of cartilage by regulating CRB3, so FBP1 is a potential target in treating OA.

Diamond controls epithelial polarity through the dynactin-dynein complex.

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Here, using Drosophila ovarian follicular epithelia as a model, we show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus our data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity.

Border cell polarity and collective migration require the spliceosome component Cactin.

Border cells are an in vivo model for collective cell migration. Here, we identify the gene cactin as essential for border cell cluster organization, delamination, and migration. In Cactin-depleted cells, the apical proteins aPKC and Crumbs (Crb) become abnormally concentrated, and overall cluster polarity is lost. Apically tethering excess aPKC is sufficient to cause delamination defects, and relocalizing apical aPKC partially rescues delamination. Cactin is conserved from yeast to humans and has been implicated in diverse processes. In border cells, Cactin's evolutionarily conserved spliceosome function is required. Whole transcriptome analysis revealed alterations in isoform expression in Cactin-depleted cells. Mutations in two affected genes, Sec23 and Sec24CD, which traffic Crb to the apical cell surface, partially rescue border cell cluster organization and migration. Overexpression of Rab5 or Rab11, which promote Crb and aPKC recycling, similarly rescues. Thus, a general splicing factor is specifically required for coordination of cluster polarity and migration, and migrating border cells are particularly sensitive to splicing and cell polarity disruptions.

Identification and characterization of Crumbs polarity complex proteins in Caenorhabditis elegans

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.

Multiple PDZ domain protein maintains patterning of the apical cytoskeleton in sensory hair cells.

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here, we report a role for multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis in mouse. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprint proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex.

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination.

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. Here, we show that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. We show that MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. Our data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.

Crumbs proteins stabilize the cone mosaics of photoreceptors and improve vision in zebrafish

Although the unique organization of vertebrate cone mosaics was first described long ago, both their underlying molecular basis and physiological significance are largely unknown. Here, we demonstrate that Crumbs proteins, the key regulators of epithelial apical polarity, establish the planar cellular polarity of photoreceptors in zebrafish. Via heterophilic Crb2a-Crb2b interactions, the apicobasal polarity protein Crb2b restricts the asymmetric planar distribution of Crb2a in photoreceptors. The planar polarized Crumbs proteins thus balance intercellular adhesions and tension between photoreceptors, thereby stabilizing the geometric organization of cone mosaics. Notably, loss of Crb2b in zebrafish induces a nearsightedness-like phenotype in zebrafish accompanied by an elongated eye axis and impairs zebrafish visual perception for predation. These data reveal a detailed mechanism for cone mosaic homeostasis via previously undiscovered apical-planar polarity coordination and propose a pathogenic mechanism for nearsightedness.

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube.

ABSTRACTUsing the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Mpp5a- and Rab11a-mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose that these roles for both Mpp5a and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction-remodelling role of Mpp5a is not specific to dividing cells.

A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

The extracellular and intracellular regions of Crb2a play distinct roles in guiding the formation of the apical zonula adherens

The transmembrane protein Crumbs (Crb), a key regulator of apical polarity, has a known involvement in establishment of the apical zonula adherens in epithelia, although the precise mechanism remains elusive. The zonula adherens are required to maintain the integrity and orderly arrangement of epithelia. Loss of the zonula adherens leads to morphogenetic defects in the tissues derived from epithelium. In this study, we revealed that the intracellular tail of Crb2a promoted the apical distribution of adherens junctions (AJs) in zebrafish retinal and lens epithelia, but caused assembly into unstable punctum adherens-like adhesion plaques. The extracellular region of Crb2a guided the transformation of AJs from the punctum adherens into stable zonula adherens. Accordingly, a truncated form of Crb2a lacking the extracellular region (Crb2aΔEX) could only partially rescue the retinal patterning defects in crb2a null mutant zebrafish (crb2am289). By contrast, constitutive over-expression of Crb2aΔEX disrupted the integrity of the outer limiting membrane in photoreceptors, which is derived from the zonula adherens of the retinal neuroepithelium. This study demonstrated that both the extracellular region and the intracellular tail of Crb2a are required to guide the formation of the apical zonula adherens.

Good Fences Make Good Neighbors: Crumbs Regulates Rho-Kinase Dynamics to Assemble a Tissue Boundary.

Boundary formation between nascent tissues prevents cell mixing, powering morphogenesis. In this issue of Developmental Cell, Sidor etal. (2020) describe a novel mechanism whereby the homophilic adhesion protein Crumbs regulates planar-polarized assembly of actomyosin cables at tissue boundaries by affecting dynamics of membrane recruitment of the myosin regulator Rho-kinase.

Rho-Kinase Planar Polarization at Tissue Boundaries Depends on Phospho-regulation of Membrane Residence Time

SummaryThe myosin II activator Rho-kinase (Rok) is planar polarized at the tissue boundary of the Drosophila embryonic salivary gland placode through a negative regulation by the apical polarity protein Crumbs that is anisotropically localized at the boundary. However, in inner cells of the placode, both Crumbs and Rok are isotropically enriched at junctions. We propose that modulation of Rok membrane residence time by Crumbs’ downstream effectors can reconcile both behaviors. Using FRAP combined with in silico simulations, we find that the lower membrane dissociation rate (koff) of Rok at the tissue boundary with low Crumbs explains this boundary-specific effect. The S/T kinase Pak1, recruited by Crumbs and Cdc42, negatively affects Rok membrane association in vivo and in vitro can phosphorylate Rok near the pleckstrin homology (PH) domain that mediates membrane association. These data reveal an important mechanism of the modulation of Rok membrane residence time via affecting the koff that may be widely employed during tissue morphogenesis.

Crumbs proteins regulate layered retinal vascular development required for vision

Crumbs proteins are transmembrane proteins that regulate cellular apico-basal polarity. Animals carrying mutated crb1 present retinal vascular abnormalities; this mutation is associated with progressive retinal degeneration with intraretinal cystoid fluid collection in humans. This study aimed to evaluate a potential role of crumbs proteins in retinal vascular development and maintenance. We demonstrated that crumbs homologues (CRBs) were differentially expressed and changed dramatically during mouse retinal vascular development. Intravitreal injection of CRB1 and CRB2 siRNA induced delayed development of the deep capillary plexus and premature development of the intermediate capillary plexus, resulting in disrupted vascular integrity. However, microfluidic chip assay using human retinal endothelial cells revealed that CRBs do not directly affect in vitro retinal angiogenesis. CRBs control retinal angiogenesis by regulating neuroglial vascular endothelial growth factor-A (VEGFA) and matrix metalloproteinase-3 expression. These findings demonstrate a pivotal role of CRBs in providing critical neurotrophic support through normal layered vascular network development and maintenance. This implies that preserving CRBs and restoring layered retinal vascular networks could be novel targets for preventing vision-threatening retinal diseases.

Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila.

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Casein kinase 1 family proteins promote Slimb-dependent Expanded degradation.

Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/β-TrCP SCF E3 ligase complex. Here, we show that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/β-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.

Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction.

During morphogenesis, epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key for the localisation of the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. Here, we applied quantitative fluorescence techniques to show that, during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage-dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain-binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin (also known as Shotgun) turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity.This article has an associated First Person interview with the first author of the paper.

Crumbs protein homolog 3 (CRB3) expression is associated with oestrogen and progesterone receptor positivity in breast cancer.

The crumbs protein homolog 3 (CRB3) regulates the tight junction to help maintain epithelial polarity. Altered CRB3 expression was associated with carcinogenesis of epithelial cells. This study detected CRB3 expression in 192 cases of breast cancer tissues and in the Molecular Taxonomy of Breast Cancer International Consortium (Metabric) and The Cancer Genome Atlas (TCGA) datasets for association with triple negative breast cancer (TNBC) phenotypes. The in vitro experiments confirm the ex vivo data. The data showed that levels of both CRB3 mRNA and protein were associated with TNBC phenotypes, ie, 41.1% (39/95) of ER+ breast cancer was CRB3‐positive, whereas 26.9% (25/93) ER– tumour was CRB3‐positive (P = 0.046). Moreover, 47.6% (30/63) of PR+ breast cancer was CRB3‐positive vs 28.4% (33/116) PR‐ tumours positive for CRB3 (P = 0.013). In addition, 40.1% (27/66) of ER+/PR+ tumour was CRB3‐positive, but only 22.4% (19/85) of TNBC showed CRB3 expression (P = 0.048). Indeed, levels of CRB3 mRNA were higher in non‐TNBC than TNBC in both Metabric (P = 3.682e‐10) and TCGA datasets (P = 2.501e‐07). The in vitro data showed that CRB3 expression was higher in luminal (MCF7 and T47D) than in HER2 (MDA‐MB‐453 and SK‐BR‐3) and basal (MDA‐MB‐231 and BT‐549) breast cancer cell lines. More interestingly, ERα regulated expression of CRB3 protein in MCF7 and BT‐549 cells and ERα expression was associated with CRB3 expression in breast cancer tissues specimens. This study demonstrated that ERα could be a novel regulator for CRB3 expression in breast cancer.