Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response.

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

The 3'UTR of the α6 integrin message regulates localization of α6ß4 integrin heterodimers.

The α6ß4 integrin heterodimer is an essential component of hemidesmosomes (HDs) and HD-related structures, which adhere epithelial cells to the underlying extracellular matrix. In this study, we focused on the importance of the α6 integrin 3' untranslated region (UTR) in α6ß4 integrin localization. To do so, A549 cells (a type II lung alveolar cell line) and immortalized human epidermal keratinocytes (iHEK) were infected with adenovirus encoding the entire α6 integrin protein with or without portions of its 3'UTR. In infected A549 cells, we detected α6ß4 integrin heterodimers containing the product of the adenovirus, regardless of whether the α6 integrin 3'UTR was present. However, only those α6 integrin proteins whose messages contained bases 4770-5633 of the α6 integrin 3'UTR were targeted to matrix adhesion sites. Moreover, overexpression of the full length α6 integrin 3'UTR, minus the coding sequence, in A549 cells disrupts the localization of endogenous α6ß4 integrin heterodimers. Following infection of iHEKs with the same adenovirus, the induced α6 integrin protein localizes to HDs regardless of whether its message possessed a 3'UTR. In sharp contrast, in α6 integrin depleted iHEKs, restoring α6 integrin expression using the coding sequence alone via adenoviral transduction resulted in α6 integrin preferentially forming α6ß1 rather than α6ß4 integrin heterodimers. α6ß4 integrin was only observed in knocked down cells following infection of adenovirus encoding the α6 integrin coding sequence with its 3'UTR. In summary, our data indicate that the α6 integrin 3'UTR is a key regulator of α6ß4 integrin heterodimer assembly and incorporation at sites of cell-matrix adhesion.

Nesprin-2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces.

The nuclear envelope protein nesprin-2G is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex and is responsible for mechanical and signaling crosstalk between the nucleus and cytoskeleton. A prior study has demonstrated that nesprin-2G knockout mice show delayed wound healing. The goal was to elucidate the mechanism underlying the delayed wound closure in this mouse model. Primary fibroblasts from wild-type and knockout neonatal mice were isolated. Knockout cells exhibited decreased focal adhesion (FA) size, number, and intensity. Consistent with this result, FA protein expression levels were decreased in knockout cells. Additionally, knockout fibroblasts displayed an abnormal actin cytoskeleton, as evidenced by loss of TAN line formation and both cytoplasmic and peri-nuclear actin staining. Using collective and single cell motility assays, it was found that knockout cells exhibited a reduction in both speed and directed migration. Traction force microscopy revealed that knockout fibroblasts generated fewer traction forces compared with WT fibroblasts. In summary, the data indicated that changes in actin organization and defects in FAs result in a reduced ability of knockout fibroblasts to generate traction forces needed for efficient motility.

Complexes of α6ß4 integrin and vimentin act as signaling hubs to regulate epithelial cell migration.

We find that clusters of ß4 integrin, organized into distinct puncta, localize along vimentin filaments within lamellipodia at the cell edge of A549 cells, as assessed by interferometric photoactivated localization microscopy. Moreover, puncta and vimentin filaments exhibit a dynamic interplay in live cells, as viewed by structured-illumination microscopy, with ß4 integrin puncta that associate with vimentin persisting for longer than those that do not. Interestingly, in A549 cells ß4 integrin regulates vimentin cytoskeleton organization. When ß4 integrin is knocked down there is a loss of vimentin filaments from lamellipodia. However, in these conditions, vimentin filaments instead concentrate around the nucleus. Although ß4 integrin organization is unaffected in vimentin-deficient A549 cells, such cells move in a less-directed fashion and exhibit reduced Rac1 activity, mimicking the phenotype of ß4 integrin-deficient A549 cells. Moreover, in vimentin-deficient cells, Rac1 fails to cluster at sites enriched in α6ß4 integrin heterodimers. The aberrant motility of both ß4 integrin and vimentin-deficient cells is rescued by expression of active Rac1, leading us to propose that complexes of ß4 integrin and vimentin act as signaling hubs, regulating cell motility behavior.

Loss of ß-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation.

During healing of the skin, the cytoskeleton of keratinocytes and their matrix adhesions, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by small GTPases and their regulators, including the guanine nucleotide exchange factor ß-PIX (also known as ARHGEF7). In fibroblasts, ß-PIX activates small GTPases, thereby enhancing migration. In keratinocytes in vitro, ß-PIX localizes to FAs. To study ß-PIX functions, we generated ß-PIX knockdown keratinocytes. During wound closure of ß-PIX knockdown cell monolayers, disassembly of FAs is impaired, and their number and size are increased. In addition, in the ß-PIX knockdown cells, phosphorylated myosin light chain (MLC; also known as MYL2) is present not only in the leading edge of cells at the wound front, but also in the cells following the front, while p21-activated kinase 2 (PAK2), a regulator of MLC kinase (MYLK), is mislocalized. Inhibition or depletion of MYLK restores FA distribution in ß-PIX knockdown cells. Traction forces generated by ß-PIX knockdown cells are increased relative to those in control cells, a result consistent with an unexpected enhancement in the migration of single ß-PIX knockdown cells and monolayers of such cells. We propose that targeting ß-PIX might be a means of promoting epithelialization of wounds in vivo.

Intermediate Filaments and the Plasma Membrane.

A variety of intermediate filament (IF) types show intricate association with plasma membrane proteins, including receptors and adhesion molecules. The molecular basis of linkage of IFs to desmosomes at sites of cell-cell interaction and hemidesmosomes at sites of cell-matrix adhesion has been elucidated and involves IF-associated proteins. However, IFs also interact with focal adhesions and cell-surface molecules, including dystroglycan. Through such membrane interactions, it is well accepted that IFs play important roles in the establishment and maintenance of tissue integrity. However, by organizing cell-surface complexes, IFs likely regulate, albeit indirectly, signaling pathways that are key to tissue homeostasis and repair.

α6ß4 Integrin Regulates the Collective Migration of Epithelial Cells.

α6ß4 integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of ß4 integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α6 integrin retains its punctate distribution. Moreover, in ß4 integrin knockdown cells, we observe a loss of ß1 integrin from focal adhesions and an enhanced association with α6 integrin. We confirmed the switch in the ß integrin binding partner of α6 integrin in the knockdown cells by immunoprecipitation. We next investigated the role of ß4 integrin in collective cell migration. Wounded monolayers of ß4 integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of ß4 integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of ß4 integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which ß4 integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from ß4 integrin deficiency. Together, our results support the hypothesis that α6ß4 integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.

Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.

Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration.

Pre- and Post-embedding Immunogold Labeling of Tissue Sections.

Despite the improved resolution capacities of fluorescence microscopy over the last 20 years, localization of specific proteins at the ultrastructural level with gold-conjugated antibodies remains a valuable technique in the cell biological tool chest. Ultrastructural immunolocalization of specific proteins in tissues rather than in cultured cells is often advantageous because, in tissues, the interactions between different cell types and with the extracellular matrix are maintained. Here, we describe two immunogold labeling procedures to localize at the ultrastructural level one or more proteins. In the first procedure (pre-embedding), micrometer-thick tissue cryostat sections are immunostained prior to embedding for obtaining ultrathin sections suitable for TEM, while in the second procedure (post-embedding), tissues are embedded in a hydrophobic resin such as Lowicryl K4M and ultrathin sections are first obtained and then immunolabeled. While the former method is better at generating strong immunolabeling, the latter is better at preserving ultrastructure.

Focusing super resolution on the cytoskeleton.

Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.

A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.-Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and ß4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration.

Lung-specific loss of α3 laminin worsens bleomycin-induced pulmonary fibrosis.

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-ß was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-ß. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

A new component of the Fraser complex.

In embryos, the Fraser complex (FC) mediates epithelial-connective tissue interactions. Loss of expression of FC components leads to Fraser syndrome (FS), in which cohesion of epithelial tissues and stroma is perturbed. Using zebrafish, Richardson et al. (this issue) identified the protein AMACO in the FC. We discuss the utility of zebrafish in determining FC functions and identifying FS targets.

Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair.

Significance: During wound healing of the skin, keratinocytes should move over while still adhering to their underlying matrix. Thus, mechanistic insights into the wound-healing process require an understanding of the forms and functions of keratinocyte matrix adhesions, specifically focal contacts and hemidesmosomes, and their components. Recent Advances: Although the structure and composition of focal contacts and hemidesmosomes are relatively well defined, the functions of their components are only now being delineated using mouse genetic models and knockdown approaches in cell culture systems. Remarkably, both focal contact and hemidesmosomal proteins appear involved in determining the speed and directional migration of epidermal cells by modulating several signal transduction pathways. Critical Issues: Although many publications are centered on focal contacts, their existence in tissues such as the skin is controversial. Nonetheless, focal contact proteins are central to mechanisms that regulate skin cell motility. Conversely, hemidesmosomes have been identified in intact skin but whether hemidesmosomal components play a positive regulatory function in keratinocyte motility remains debated in the field. Future Directions: Defective wound healing is a developing problem in the aged, hospitalized and diabetic populations. Hence, deriving new insights into the molecular roles of matrix adhesion proteins in wound healing is a prerequisite to the development of novel therapeutics to enhance tissue repair and regeneration.

A role for anti-BP180 autoantibodies in chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Chronic Rhinosinusitis (CRS) is accompanied by evidence of a vigorous adaptive immune response, and emerging studies demonstrate that some nasal polyps manifest a polyclonal autoantibody response. We previously found that antibodies against BP180, a component of the hemidesmosome complex and the dominant epitope in autoimmune bullous pemphigoid, were found at elevated levels in nasal polyp tissue. Given the critical role of hemidesmosomes in maintaining epithelial integrity, we sought to investigate the distribution of BP180 in nasal tissue and evaluate for evidence of systemic autoimmunity against this antigen in CRS. STUDY DESIGN: Case-control experimental study. METHODS: The expression and distribution of BP180 in cultured nasal epithelial cells and normal nasal tissue were confirmed using real-time polymerase chain reaction (PCR), Western immunoblotting, immunofluorescence and immunohistochemistry. Sera were collected from three groups: control, CRSsNP, and CRSwNP. A commercially available ELISA was utilized to compare anti-BP180 autoantibody levels in sera. RESULTS: BP180 is expressed in nasal epithelium, but is not confined to the basement membrane as it is in human skin. In cultured nasal epithelial cells, confocal immunofluorescence showed a punctate distribution of BP180 along the basal surface, consistent with its distribution in epithelial keratinocytes. There are significantly higher levels of circulating nonpathologic anti-BP180 autoantibodies in CRS patients compared with normal controls (P <0.05). CONCLUSIONS: BP180 is more widely expressed in nasal epithelium versus skin, although it appears to play a similar role in the formation of hemidesmosomes along the basement membrane. Further investigations are ongoing to characterize the pathogenicity of the anti-epithelial antibody response in CRS.

Plectin-containing, centrally localized focal adhesions exert traction forces in primary lung epithelial cells.

Receptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.

Laminin-332 and α3ß1 integrin-supported migration of bronchial epithelial cells is modulated by fibronectin.

The repair of the bronchiolar epithelium damaged by cell-mediated, physical, or chemical insult requires epithelial cell migration over a provisional matrix composed of complexes of extracellular matrix molecules, including fibronectin and laminin. These matrix molecules support migration and enhance cell adhesion. When cells adhere too tightly to their matrix they fail to move; but if they adhere too little, they are unable to develop the traction force necessary for motility. Thus, we investigated the relative contributions of laminin and fibronectin to bronchiolar cell adhesion and migration using the immortalized bronchial lung epithelial cell line (BEP2D) and normal human bronchial epithelial (NHBE) cells, both of which assemble a laminin α3ß3γ2 (LM332)/fibronectin-rich matrix. Intriguingly, BEP2D and NHBE cells migrate significantly faster on an LM332-rich matrix than on fibronectin. Moreover, addition of fibronectin to LM332 matrix suppresses motility of both cell types. Finally, fibronectin enhances the adhesion of both BEP2D and NHBE cells to LM332-coated surfaces. These results suggest that fibronectin fine tunes LM332-mediated migration by boosting bronchiolar cell adhesion to substrate. We suggest that, during epithelial wound healing of the injured airway, fibronectin plays an important adhesive role for laminin-driven epithelial cell motility by promoting a stable cellular interaction with the provisional matrix.

Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease induced by pathogenic autoantibodies against a type II transmembrane protein (BP180, collagen type XVII, or BPAG2). In animal models, BP180 autoantibody-antigen interaction appears insufficient to develop blisters, but involvement of complement and neutrophils is required. However, cultured keratinocytes treated with BP-IgG exhibit a reduction in the adhesive strength and a loss of expression of BP180, suggesting that the autoantibodies directly affect epidermal cell-extracellular matrix integrity. In this study, we explored the consequences of two distinct epithelial cells treated with BP-IgG, particularly the fate of BP180. First, we followed the distribution of green fluorescent protein-tagged BP180 in an epithelial cell line, 804G, and normal human epidermal keratinocytes after autoantibody clustering. After BP-IgG treatment, the adhesive strength of the cells to their substrate was decreased, and BP180 was internalized in both cell types, together with the early endosomal antigen-1. By using various endocytosis inhibitors and a fluid-uptake assay, we demonstrated that BP-IgG-induced BP180 internalization is mediated via a macropinocytic pathway. Moreover, a macropinocytosis inhibitor rescued a BP-IgG-induced reduction in the adhesive strength of the cells from their substrate. The results of this study suggest that BP180 internalization induced by BP-IgG plays an important role in the initiation of disease pathogenesis.

Actinin-4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions.

During wound repair, epidermal cells at the edge of an injury establish front-rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin-4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin-4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin-4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin-332 matrix. In actinin-4-knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6ß4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin-4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.