Defining the Insect Pollinator Community Found in Iowa Corn and Soybean Fields: Implications for Pollinator Conservation.

Although corn (Zea mays L.) and soybeans (Glycine max L.) do not require pollination, they offer floral resources used by insect pollinators. We asked if a similar community of insect pollinators visits these crops in central Iowa, a landscape dominated by corn and soybean production. We used modified pan traps (i.e., bee bowls) in both corn and soybean fields during anthesis and used nonmetric multidimensional scaling (NMS) to compare the communities found in the two crops. Summed across both crops, 6,704 individual insects were captured representing at least 60 species, morphospecies, or higher-level taxa. Thirty-four species were collected in both crops, 19 collected only in corn and seven were collected only in soybean. The most abundant taxa were Lasioglossum [Dialictus] spp., Agapostemon virescens Cresson, Melissodes bimaculata (Lepeletier), and Toxomerus marginatus (Say), which accounted for 65% of the insect pollinators collected from both crops. Although social bees (Apis mellifera L. and Bombus spp.) were found in both crops, they accounted for only 0.5% of all insects captured. The NMS analysis revealed a shared community of pollinators composed of mostly solitary, ground nesting bees. Many of these species have been found in other crop fields throughout North America. Although corn and soybean are grown in landscapes that are often highly disturbed, these data suggest that a community of pollinators can persist within them. We suggest approaches to conserving this community based on partnering with activities that aim to lessen the environmental impact of annual crop production.

Insect Pollinators in Iowa Cornfields: Community Identification and Trapping Method Analysis.

Availability of mass flowering plants in landscapes dominated by agriculture can have a strong positive impact on the density of generalist, native pollinators. Row-crop production in Iowa accounts for 75% of the arable acres, with corn, Zea mays, representing the majority of hectares planted. To date, there has been no description of the insect pollinator community found within Iowa cornfields. We report a field study to determine the optimal sampling methodology to characterize the community of insect pollinators within cornfields. During 2012 and 2013, 3,616 insect pollinators representing 51 species were captured using bee bowls, and 945 individuals representing 10 species were captured using sticky cards. We examined the effects of trap type, height, and bowl color on the described community. Bee bowls captured a more abundant and species rich community than sticky cards with all species captured on sticky cards also present in bee bowls. Traps deployed at the height of the tassels describe a more abundant and species rich community of pollinators than traps at ear height (2x as many individuals) or ground height (4x as many individuals). Blue bowls captured more bees than white (2.75x as many individuals) or yellow bowls (3.5x as many individuals); and yellow bowls captured more flies than white (2x as many individuals) or blue (2.3x as many individuals). To provide the most complete description of the community of insect pollinators using cornfields as a resource, we suggest sampling-using bee bowls at the height of the tassels using all three bee bowl colors.

Expression of inappropriate cadherins by epithelial tumor cells promotes endocytosis and degradation of E-cadherin via competition for p120(ctn).

Cadherin cell-cell adhesion proteins play an important role in modulating the behavior of tumor cells. E-cadherin serves as a suppressor of tumor cell invasion, and when tumor cells turn on the expression of a non-epithelial cadherin, they often express less E-cadherin, enhancing the tumorigenic phenotype of the cells. Here, we show that when A431 cells are forced to express R-cadherin, they dramatically downregulate the expression of endogenous E- and P-cadherin. In addition, we show that this downregulation is owing to increased turnover of the endogenous cadherins via clathrin-dependent endocytosis. p120(ctn) binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120(ctn) may accomplish this is to serve as a rheostat to regulate the levels of cadherin. Here, we show that the degradation of E-cadherin in response to expression of R-cadherin is owing to competition for p120(ctn).

Cadherin junctions in mammary tumors.

Cadherins are the transmembrane component of adherens junctions found between interacting cells in tissues. The cadherins bind cells to one another in a specific manner and link to the actin cytoskeleton through intracellular catenins. In addition to promoting strong cell-cell adhesion, cadherins appear to initiate and modify intracellular signaling pathways. The loss of E-cadherin function in epithelial cells is thought to be an important step in tumorigenesis. Moreover, anomalous expression of inappropriate cadherins in epithelial cells alters their behavior and may contribute to the tumorigenic phenotype. For breast cancer the decreased expression of E-cadherin alone may have limited value as a prognostic indicator; however, examining the repertoire of cadherins and catenins expressed by tumors may provide useful prognostic information.

Upregulation of P-cadherin expression in the lesional skin of pemphigus, Hailey-Hailey disease and Darier's disease.

BACKGROUND: Autoimmune blistering diseases, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), are known to be caused by binding of autoantibodies to the desmosomal cadherins, desmoglein 3 and desmoglein 1, respectively. Recently, mutations in the genes coding Ca2+ pumps leads to inherited blistering diseases, Hailey-Hailey disease (HHD) and Darier's disease (DD). Cadherins are a family of Ca2+-dependent cell adhesion molecules and P-cadherin is one of the major cadherins expressed in the epidermis. Although detailed mechanisms of acantholysis of these blistering diseases have not been fully clarified, abnormal expression of cadherins caused by altered Ca2+ concentration due to the binding of autoantibodies to cell surface or by mutations in Ca2+ pumps is suggested to be involved in mechanisms of acantholysis of these autoimmune and inherited blistering diseases. The purpose of the present study was to determine whether altered P-cadherin expression is present in these diseases. METHOD: Distribution patterns of P-cadherin in skin specimens from patients with PV (n=2), PF (n=2), HHD (n=4) and DD (n=3), were examined with confocal laser scanning microscopy using two anti-P-cadherin antibodies, 6A9 and NCC-CAD-299. RESULTS: In normal control skin, P-cadherin expression was restricted to the basal layer. In contrast, positive immunostaining of P-cadherin was observed not only in the basal cells, but also in the suprabasal cells in lesional skin of all the acantholytic diseases. CONCLUSIONS: The present results clearly demonstrated that upregulation of P-cadherin expression occurs in the acantholysis in all the four blistering diseases PV, PF, HHD and DD. Upregulation of P-cadherin may be involved in the pathomechanism of both the autoimmune blistering diseases and the inherited blistering diseases.

N-cadherin is developmentally regulated and functionally involved in early hematopoietic cell differentiation.

The cadherins, an important family of cell adhesion molecules, are known to play major roles during embryonic development and in the maintenance of solid tissue architecture. In the hematopoietic system, however, little is known of the role of this cell adhesion family. By RT-PCR, western blot analysis and immunofluorescence staining we show that N-cadherin, a classical type I cadherin mainly expressed on neuronal, endothelial and muscle cells, is expressed on the cell surface of resident bone marrow stromal cells. FACS analysis of bone marrow mononuclear cells revealed that N-cadherin is also expressed on a subpopulation of early hematopoietic progenitor cells. Triple-color FACS analysis defined a new CD34(+) CD19(+) N-cadherin(+) progenitor cell population. During further differentiation, however, N-cadherin expression is lost. Treatment of CD34(+) progenitor cells with function-perturbing N-cadherin antibodies drastically diminished colony formation, indicating a direct involvement of N-cadherin in the differentiation program of early hematopoietic progenitors. N-cadherin can also mediate adhesive interactions within the bone marrow as demonstrated by inhibition of homotypic interactions of bone-marrow-derived cells with N-cadherin antibodies. Together, these data strongly suggest that N-cadherin is involved in the development and retention of early hematopoietic progenitors within the bone marrow microenvironment.

Truncated DCC reduces N-cadherin/catenin expression and calcium-dependent cell adhesion in neuroblastoma cells.

The deleted in colorectal cancer (DCC) protein is important in the pathway guidance of cells and cell processes during neural development, and DCC has also been implicated in the aberrant cellular migrations of neuroblastoma dissemination. We attempted to further define DCC protein function by the overexpression of full-length and truncated DCC constructs in a human neuroblastoma cell line. Overexpression of the truncated DCC protein resulted in a less epithelioid morphology. This was accompanied by decreases in expression of N-cadherin and alpha- and beta-catenin by immunoblot and Northern blot analysis. Levels of desmoglein were relatively less affected, whereas endogenous DCC protein levels were increased in the truncated transfectants. N-cadherin immunofluorescence was consistent with the immunoblot studies and localized the protein to the cytoplasm and sites of cell-cell contact. Cell aggregation studies demonstrated diminished calcium-dependent aggregation in the truncated transfectants. In conclusion, overexpression of a truncated DCC protein in neuroblastoma cells resulted in the loss of an epithelioid morphology, diminished expression of N-cadherin and alpha- and beta-catenin, and diminished calcium-dependent cell adhesion. These studies provide the first evidence of an apparent functional link between DCC and N-cadherin/catenin-dependent cell adhesion.

N-Cadherin extracellular repeat 4 mediates epithelial to mesenchymal transition and increased motility.

E- and N-cadherin are members of the classical cadherin family of proteins. E-cadherin plays an important role in maintaining the normal phenotype of epithelial cells. Previous studies from our laboratory and other laboratories have shown that inappropriate expression of N-cadherin by tumor cells derived from epithelial tissue results in conversion of the cell to a more fibroblast-like cell, with increased motility and invasion. Our present study was designed to determine which domains of N-cadherin make it different from E-cadherin, with respect to altering cellular behavior, such as which domains are responsible for the epithelial to mesenchymal transition and increased cell motility and invasion. To address this question, we constructed chimeric cadherins comprised of selected domains of E- and N-cadherin. The chimeras were transfected into epithelial cells to determine their effect on cell morphology and cellular behavior. We found that a 69-amino acid portion of EC-4 of N-cadherin was necessary and sufficient to promote both an epithelial to mesenchymal transition in squamous epithelial cells and increased cell motility. Here, we show that different cadherin family members promote different cellular behaviors. In addition, we identify a novel activity that can be ascribed to the extracellular domain of N-cadherin.

Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer.

Breast cancers often show reduced expression of the transmembrane cell-cell adhesion protein, E-cadherin. In addition, approximately half of breast carcinomas express P-cadherin, which correlates with poor survival. A large fragment of the E-cadherin extracellular domain can be detected in serum, and it has been proposed that an increase in serum E-cadherin can denote the presence of a tumor. In this study, we tested the possibility that serum E- or P-cadherin levels might be useful diagnostic or prognostic indicators in breast cancer. However, we found no indication that the level of serum E-cadherin correlated with the presence of breast cancer. In addition, although we successfully detected a fragment of P-cadherin in serum, we found that its level was considerably lower than that of E-cadherin and did not correlate with the presence of P-cadherin-positive breast cancer.

Dissociation of intra- and extracellular domains of desmosomal cadherins and E-cadherin in Hailey-Hailey disease and Darier's disease.

In order to clarify the pathomechanism of acantholysis in Hailey-Hailey disease (HHD) and Darier's disease (DD), the distribution of desmosomal and adherens junction-associated proteins was studied in the skin of patients with HHD (n = 4) and DD (n = 3). Domain-specific antibodies were used to determine the cellular localization of the desmosomal transmembrane glycoproteins (desmogleins 1 and 3 and desmocollin), desmosomal plaque proteins (desmoplakin, plakophilin and plakoglobin) and adherens junction-associated proteins (E-cadherin, alpha-catenin, beta-catenin and actin). A significant difference in staining patterns between intra- and extracellular domains of desmosomal cadherins and E-cadherin was demonstrated in acantholytic cells in both HHD and DD, but not in those in pemphigus vulgaris and pemphigus foliaceus samples used as controls. In acantholytic cells in HHD and DD, antibodies against attachment plaque proteins and intracellular epitopes of desmosomal cadherins exhibited diffuse cytoplasmic staining, whereas markedly reduced staining was observed with antibodies against extracellular epitopes of the desmogleins. Similarly, membrane staining of an intracellular epitope of E-cadherin was preserved, while immunoreactivity of an extracellular epitope of E-cadherin was destroyed. While the DD gene has been identified as ATP2A2, the gene for HHD has not been clarified. The dissociation of intra- and extracellular domains of desmosomal cadherin and E-cadherin is characteristic of the acantholytic cells in HHD and DD, and not of pemphigus. This common phenomenon in HHD and DD might be closely related to the pathophysiological mechanisms in both conditions.

Vimentin expression in human squamous carcinoma cells: relationship with phenotypic changes and cadherin-based cell adhesion.

Phenotypic changes resembling an epithelial-to-mesenchymal transition often occur as epithelial cells become tumorigenic. Two proteins that have been implicated in this process are vimentin and N-cadherin. In this study, we sought to establish a link between expression of vimentin and N-cadherin as oral squamous epithelial cells undergo a morphologic change resembling an epithelial-to-mesenchymal transition. We found that N-cadherin and vimentin did not influence the expression of one another.

The amino- and carboxyl-terminal tails of (beta)-catenin reduce its affinity for desmoglein 2.

beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction beta-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. beta-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of beta-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between beta-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of beta-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both beta-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of beta-catenin and propose that a sequence in the first 26 amino acids of beta-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.

Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.

Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution.

A potential target of hormone action during prostate and mammary involution is the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In a previous study addressing this aspect of involution, we acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion initiates apoptotic programs during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in cell death following apoptotic stimuli. Loss of cell-cell adhesion in the nonaggregated population appeared to result from the rapid truncation within the cytosolic domain of the mature, 120-kDa species of E-cadherin (E-cad(120)). Immunoprecipitations from cell culture and involuting mammary gland demonstrated that this truncation removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin, resulting in a non-beta-catenin binding, membrane-bound 97-kDa species (E-cad(97)) and a free cytoplasmic 35-kDa form (E-cad(35)) that is bound to beta-catenin. Examination of E-cadherin expression and cellular distribution during prostate and mammary involution revealed a dramatic reduction in junctional membrane staining that correlated with a similar reduction in E-cad(120) and accumulation of E-cad(97) and E-cad(35). The observation that E-cadherin was truncated during involution suggested that hormone depletion activated the same apoptotic pathway in vivo as observed in vitro. Based on these findings, we hypothesize that truncation of E-cadherin results in the loss of beta-catenin binding and cellular dissociation that may signal epithelial apoptosis during prostate and mammary involution. Thus, E-cadherin may be central to homeostatic regulation in these tissues by coordinating adhesion-dependent survival and dissociation-induced apoptosis.

Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion.

p120(ctn) is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell-cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin-p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin-deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike alpha- and beta-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin-mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin-mediated transition to tight cell-cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.

N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression.

E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent, homotypic cell-cell adhesion and plays a role in maintaining the normal phenotype of epithelial cells. Decreased expression of E-cadherin has been correlated with increased invasiveness of breast cancer. In other systems, inappropriate expression of a nonepithelial cadherin, such as N-cadherin, by an epithelial cell has been shown to downregulate E-cadherin expression and to contribute to a scattered phenotype. In this study, we explored the possibility that expression of nonepithelial cadherins may be correlated with increased motility and invasion in breast cancer cells. We show that N-cadherin promotes motility and invasion; that decreased expression of E-cadherin does not necessarily correlate with motility or invasion; that N-cadherin expression correlates both with invasion and motility, and likely plays a direct role in promoting motility; that forced expression of E-cadherin in invasive, N-cadherin-positive cells does not reduce their motility or invasive capacity; that forced expression of N-cadherin in noninvasive, E-cadherin-positive cells produces an invasive cell, even though these cells continue to express high levels of E-cadherin; that N-cadherin-dependent motility may be mediated by FGF receptor signaling; and that cadherin-11 promotes epithelial cell motility in a manner similar to N-cadherin.

Inhibition of cadherin function differentially affects markers of terminal differentiation in cultured human keratinocytes.

Cadherin function is required for normal keratinocyte intercellular adhesion and stratification. In the present study, we have investigated whether cadherin-cadherin interactions may also modulate keratinocyte differentiation, as evidenced by alterations in the levels of several differentiation markers. Confluent keratinocyte cultures, propagated in low Ca(2+) medium in which cadherins are not active, were pre-incubated with antibodies that block the function of E-cadherin and/or P-cadherin; Ca(2+ )was then elevated to 1 mM to activate the cadherins and induce differentiation. In control cultures (incubated with no antibody or with antibodies to other cell surface molecules), Ca(2+) elevation induced an increase in type 1 transglutaminase, profilaggrin, and loricrin, as measured by western blotting and in agreement with previous results. However, the concurrent addition of antibodies against both E- and P-cadherin prevented this increase in transglutaminase 1 protein. Incubation with either antibody alone had no consistent effect. Profilaggrin and loricrin, which are later markers of keratinocyte differentiation, responded differently from transglutaminase 1 to addition of antibodies. In the presence of anti-E-cadherin antibody, both loricrin and profilaggrin levels were dramatically enhanced compared to the high Ca(2+) control cells, while addition of antibody to P-cadherin slightly attenuated the Ca(2+)-induced increase. In the presence of both antibodies, loricrin and profilaggrin protein levels were intermediate between those observed in the presence of either antibody alone. The expression of involucrin, however, was unaffected by addition of antibodies. In addition, effects of the anti-cadherin antibodies were not secondary to alterations in proliferation or programmed cell death, as determined by several independent assays of these processes. Thus, the consequences of cadherin inhibition depend upon both the particular cadherin and the differentiation marker under study. Taken together, these data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal differentiation in the epidermis in multiple ways.

Cadherin and catenin expression in normal human bronchial epithelium and non-small cell lung cancer.

Cadherins are transmembrane cell adhesion molecules (CAMS) that mediate cell-cell interactions and are important for maintenance of epithelial cell integrity. This function is dependent on an indirect interaction between the cytoplasmic domain of the cadherin molecule with three cytoplasmic proteins known as alpha-, beta-, and gamma-catenin (-cat). Growing evidence suggests that alterations in cadherin or catenin expression or function may be important to the development of an invasive or metastatic phenotype. Immunohistochemical techniques were used to study the expression of the two major epithelial cadherins, E-cadherin (E-cad) and P-cadherin (P-cad) as well as alpha- and gamma-cat in normal bronchial epithelium and in a series of carefully TMN-staged pulmonary adenocarcinomas (n = 21) and squamous cell carcinomas (n = 7). The cadherin profile of normal pseudostratified bronchial epithelium was heterogeneous. Basilar cells strongly expressed P-cad, alpha- and gamma-cat, while columnar cells moderately expressed E-cad, alpha- and gamma-cat. In contrast to other epithelial tumors, E-cad on non-small cell lung carcinomas was actually upregulated, however, a decrease in P-cad expression was noted in 68%. At least one cadherin or catenin was downregulated, compared to normal bronchial epithelium, in 82% of tumors examined. With the exception of an association between loss of P-cad expression and poorly differentiated state, changes in cadherin and catenin expression levels were not significantly correlated to tumor stage, cell type, or nodal status. These findings illustrate that alteration of expression of cadherins and catenins are often found in non-small cell lung carcinoma when compared to the progenitor bronchial epithelium, and may play a role in the development of the malignant phenotype.

Src induces morphological changes in A431 cells that resemble epidermal differentiation through an SH3- and Ras-independent pathway.

The role of Src family tyrosine kinases in cellular proliferation is well established; however, their role in cellular differentiation is less well understood. In this study we have investigated the role played by Src in the differentiation of squamous epithelial cells. Transfection of activated Src into A431 cells resulted in morphological changes that resembled epidermal differentiation. When we used Src mutants to characterize the observed phenotypic changes, we found that protein tyrosine kinase activity, correct membrane localization and the activity of the SH2 domain were required, but the SH3 domain was not. Furthermore, downstream activity of Ras was not required for the Src-mediated changes in A431 cells.