Desmoplakin controls microvilli length but not cell adhesion or keratin organization in the intestinal epithelium

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This adhesion is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, which concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. In this paper, we report that the desmosomal protein desmoplakin (DP) is not essential for cell adhesion in the intestinal epithelium. Surprisingly, when DP is lacking, keratin filament localization is also unperturbed, although keratin filaments no longer anchor at desmosomes. Unexpectedly, DP is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in simple epithelium.     

Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.     

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

The role of apical cell–cell junctions and associated cytoskeleton in mechanotransduction

Tissues of multicellular organisms are characterised by several types of specialised cell–cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton‐associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.     

An epidermal plakin that integrates actin and microtubule networks at cellular junctions

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.     

Crosstalk between Cell Adhesion Complexes in Regulation of Mechanotransduction

Physical forces regulate numerous biological processes during development, physiology, and pathology. Forces between the external environment and intracellular actin cytoskeleton are primarily transmitted through integrin-containing focal adhesions and cadherin-containing adherens junctions. Crosstalk between these complexes is well established and modulates the mechanical landscape of the cell. However, integrins and cadherins constitute large families of adhesion receptors and form multiple complexes by interacting with different ligands, adaptor proteins, and cytoskeletal filaments. Recent findings indicate that integrin-containing hemidesmosomes oppose force transduction and traction force generation by focal adhesions. The cytolinker plectin mediates this crosstalk by coupling intermediate filaments to the actin cytoskeleton. Similarly, cadherins in desmosomes might modulate force generation by adherens junctions. Moreover, mechanotransduction can be influenced by podosomes, clathrin lattices, and tetraspanin-enriched microdomains. This review discusses mechanotransduction by multiple integrin- and cadherin-based cell adhesion complexes, which together with the associated cytoskeleton form an integrated network that allows cells to sense, process, and respond to their physical 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.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. We show that, in the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and beta-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid- and Echinoid+ cells, like those between DE-Cadherin- and DE-Cadherin+ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion.     

Cell junctions during morphogenesis of feathers: general ultrastructure with emphasis on adherens junctions

Alibardi, L. 2011. Cell junctions during morphogenesis of feathers: general ultrastructure with emphasis on adherens junctions. —Acta Zoologica (Stockholm) 92 : 89–100. The present ultrastructural and immunocytochemical study analyzes the cell junctions joining barb/barbule cells versus cell junctions connecting supportive cells in forming feathers. Differently from the epidermis or the sheath, desmosomes are not the prevalent junctions among feather cells. Numerous adherens junctions, some gap junctions and fewer tight junctions are present among differentiating barb/barbule cells during early stages of their differentiation. Adherens junctions are frequent also among differentiating supportive cells and show weak immunolabeling for both N‐cadherin and neural‐cell adhesion molecule (N‐CAM). Differentiating barb and barbule cells do not show labeled junctions for N‐cadherin and N‐CAM. The labeling occurs at patches in the cytoplasm of supportive cells but is more frequently seen in the external cytoplasm and along the extra‐cellular space (glycocalix) covering the plasma membrane of supportive cells. Labeling for N‐cadherin is also found in medium‐dense 0.1‐ to 0.3‐μm granules present in supportive cells and sometimes is associated with coarse filaments or periderm granules. The study indicates that adherens junctions form most of the transitional connections among supportive cells before their degeneration. Keratinizing barb and barbule cells loose the labeling for adherens junctions (N‐CAM and N‐chaderin) while their adhesion is strengthened by the incorporation of cell junctions in the corneous mass forming the barbules.     

Cell Adhesion: Sorting out Cell Mixing with Echinoid?

Cadherins control intercellular adhesion in epithelial cells. This property relies on the ability to recruit actin filaments at adherens junctions via beta-catenin and alpha-catenin. A recent study shows that Echinoid, a member of the immunoglobulin domain containing protein family, is a modulator of intercellular adhesion in Drosophila that controls cell sorting.     

DLG-1 Is a MAGUK Similar to SAP97 and Is Required for Adherens Junction Formation

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Mathematical Modeling of the Impact of Actin and Keratin Filaments on?Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.     

Adherens junctions: new insight into assembly,modulation and function

Adherens junctions play pivotal roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling. These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells or between cells and extracellular matrix. The best-known adherens junction is the zonula adherens (ZA) that forms a belt surrounding the apical pole of epithelial cells. Recent studies in Drosophila have further illuminated the structure of adherens junctions. Scaffolding proteins encoded by the stardust gene are novel components of the Crumbs complex, which plays a critical role in ZA assembly.1-3 The small GTPase Rap1 controls the symmetric re-assembly of the ZA after cell division.4 Finally, the asymmetric distribution of adherens junction material regulates spindle orientation during asymmetric cell division in the sensory organ lineage.     

The cadherin-11 cytoplasmic juxtamembrane domain promotes alpha-catenin turnover at adherens junctions and intercellular motility

Cadherins mediate homophilic cell adhesion and contribute to tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of alpha-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 deltaJMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these data implicate the cadherin-11 cytoplasmic JMD as a regulator of alpha-catenin turnover at adherens junctions and actin-cytoskeletal organization that is critical for intercellular motility and rearrangement in multicellular clusters.     

Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry

Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.     

Rac1 functions as a reversible tension modulator to stabilize VE-cadherin trans-interaction

The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.