Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.     

Structural basis of Rho GTPase-mediated activation of the formin mDia1

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. We report the crystal structure of the dimeric regulatory domain of the DRF, mDia1. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design.     

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators during Drosophila development. We found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent.     

Adherens junctions in Drosophila retinal morphogenesis

Adherens junctions and their core molecular components, classic cadherins, make major contributions to animal morphogenesis. Although the significance of cadherins in development is generally accepted, the mechanisms regulating adherens junction function during morphogenesis remain a subject of intense research. Adherens junctions are involved in the organization of simple cellular patterns, and more complex cell shape changes and cell movements that depend on the dynamic modulation of adherens junctions.     

The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous’ role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure—migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells—as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative “fingerprint”—the protrusive profile—which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.     

DRhoGEF2 and diaphanous regulate contractile force during segmental groove morphogenesis in the Drosophila embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.     

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. Here we show that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell intercalation defects. We delineate a pathway that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, we provide evidence that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin.     

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.     

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.     

Desmosomal cadherins

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.     

Noncentrosomal microtubules and type II myosins potentiate epidermal cell adhesion and barrier formation

During differentiation, many cells reorganize their microtubule cytoskeleton into noncentrosomal arrays. Although these microtubules are likely organized to meet the physiological roles of their tissues, their functions in most cell types remain unexplored. In the epidermis, differentiation induces the reorganization of microtubules to cell–cell junctions in a desmosome-dependent manner. Here, we recapitulate the reorganization of microtubules in cultured epidermal cells. Using this reorganization assay, we show that cortical microtubules recruit myosin II to the cell cortex in order to engage adherens junctions, resulting in an increase in mechanical integrity of the cell sheets. Cortical microtubules and engaged adherens junctions, in turn, increase tight junction function. In vivo, disruption of microtubules or loss of myosin IIA and B resulted in loss of tight junction–mediated barrier activity. We propose that noncentrosomal microtubules act through myosin II recruitment to potentiate cell adhesion in the differentiating epidermis, thus forming a robust mechanical and chemical barrier against the external environment.     

FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.     

Rac function in epithelial tube morphogenesis

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.     

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.     

Rab11 Helps Maintain Apical Crumbs and Adherens Junctions in the Drosophila Embryonic Ectoderm

Background

Tissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins.

Methodology/Principal Findings

Here we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure.

Conclusions/Significance

We contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues.     

A novel role for IQGAP1 protein in cell motility through cell retraction

IQGAP1 has emerged as a key component in the regulation of cytoskeleton dynamics during cell migration, maintenance of adherens junctions, microbial pathogenesis and intracellular trafficking. IQGAP1 is known to localize to the protruding edge of lamellipodia in a variety of cell types and interact with regulators of actin dynamics. Here, we provide evidence suggesting a novel role of IQGAP1 in cell motility through cell edge retraction. In some of the cell lines examined, IQGAP1 was markedly separated from WAVE localization suggesting IQGAP1 may localize to retracting edges. B16F10 mouse melanoma cells exhibited the most restricted separation in which the appearance of GFP-IQGAP1 correlated with cell edge retraction velocity and the disappearance of mCherry-Arp3. These results demonstrate that in some cell types IQGAP1 may function to promote cell retraction not lamellipodium edge protrusion. In addition, we examined co-localization of IQGAP1 with adhesion site markers, myosin IIA, calmodulin and IQGAP2. In areas rich in IQGAP1 there was decreased immunofluorescence staining of vinculin, paxillin and phosphorylated-tyrosine indicating adhesion site disassembly. Interestingly, calmodulin, but not myosin IIA or IQGAP2, co-localized with IQGAP1 in areas of cell retraction. Overall these results suggest a new role of IQGAP1, distinct form IQGAP2, in cell migration through up regulation of contractility and downregulation of adhesion sites potentially through calmodulin interaction.     

Drosophila myosin phosphatase and its role in dorsal closure

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.