Integrin-like Proteins Research Articles

Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Transcriptomic profiling of the mussel Mytilus trossulus with a special emphasis on integrin-like genes during development

ABSTRACTThis study is based on the Illumina RNA-sequencing data obtained for a de novo assembly of the transcriptome from early developmental stages and some tissues and cells of the adult mussel Mytilus trossulus (Mytilidae, Mollusca) using the Trinity program. A total of 200,079 contigs were obtained, and compared to the NCBI database using BLAST to search for sequence similarity. The number of annotated contigs under the GO term 3 categories was estimated to reach 19.96%. The BUSCO analysis determined a level of 99.2% completeness for the assembled transcriptome. The main findings include evidence that the mussel β integrin-like protein sequences are very similar to the β integrin-like proteins so far sequenced for all classes of Mollusca, while the highest similarity is observed between mussel and oyster (Crasostrea gigas) β integrin-like proteins. Our transcriptome dataset contributes to the genetic databases of non-model animals such as bivalves and represents the first characterization of expressed sequences during early development of the mollusc M. trossulus from the Sea of Japan including the identification of candidate genes involved in cell adhesion.

Tumor endothelial marker 8 promotes cancer progression and metastasis

Every year more than 8 million people suffer from cancer-related deaths worldwide [1]. Metastasis, the spread of cancer to distant sites, accounts for 90% of these deaths. A promising target for blocking tumor progression, without causing severe side effects [2], is Tumor Endothelial Marker 8 (TEM8), an integrin-like cell surface protein expressed predominantly in the tumor endothelium and in cancer cells [3, 4]. Here, we have investigated the role of TEM8 in cancer progression, angiogenesis and metastasis in invasive breast cancer, and validated the main findings and important results in colorectal cancer. We show that the loss of TEM8 in cancer cells results in inhibition of cancer progression, reduction in tumor angiogenesis and reduced metastatic burden in breast cancer mouse models. Furthermore, we show that TEM8 regulates cancer progression by affecting the expression levels of cell cycle-related genes. Taken together, our findings may have broad clinical and therapeutic potential for breast and colorectal primary tumor and metastasis treatment by targeting TEM8.

Identification of β integrin-like- and fibronectin-like proteins in the bivalve mollusk Mytilus trossulus.

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin-like protein and its presumptive ligand, fibronectin-like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin-like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin-like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin-like protein was detected firstly at the blastula stage and later, the FN-LP-immunoreactive cells were scattered in the trochophore larvae. The fibronectin-like protein was not expressed in the β integrin-positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin- and fibronectin-like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.

The Origin and Evolution of the Plant Cell Surface: Algal Integrin-Associated Proteins and a New Family of Integrin-Like Cytoskeleton-ECM Linker Proteins.

The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed.

β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion.

Overexpression of AT14A confers tolerance to drought stress-induced oxidative damage in suspension cultured cells of Arabidopsis thaliana.

Drought stress can affect interaction between plant cell plasma membrane and cell wall. Arabidopsis AT14A, an integrin-like protein, mediates the cell wall-plasma membrane-cytoskeleton continuum (WMC continuum). To gain further insight into the function of AT14A, the role of AT14A in response to drought stress simulated by polyethylene glycol (PEG-6000) in Arabidopsis suspension cultures was investigated. The expression of this gene was induced by PEG-6000 resulting from reverse transcription-PCR, which was further confirmed by the expression data from publically available microarray datasets. Compared to the wild-type cells, overexpression of AT14A (AT14A-OE) in Arabidopsis cultures exhibited a greater ability to adapt to water deficit, as evidenced by higher biomass accumulation and cell survival rate. Furthermore, AT14A-OE cells showed a higher tolerance to PEG-induced oxidative damage, as reflected by less H2O2 content, lipid peroxidation (malondialdehyde (MDA) content), and ion leakage, which was further verified by maintaining high levels of activities of antioxidant defense enzymes such as ascorbate peroxidase and guaiacol peroxidase and soluble protein. Taken together, our results suggest that overexpression of AT14A improves drought stress tolerance and that AT14A is involved in suppressing oxidative damage under drought stress in part via regulation of antioxidant enzyme activities.

A Secretory Protein of Necrotrophic Fungus Sclerotinia sclerotiorum That Suppresses Host Resistance

SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5–3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mβCD). Removal of ergosterol by mβCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mβCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mβCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.

Arabidopsis NDR1 Is an Integrin-Like Protein with a Role in Fluid Loss and Plasma Membrane-Cell Wall Adhesion

Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, well-characterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndr1-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDR1 in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection.

Obituary: Rafael Pont-Lezica (1940?2011)

Rafael Pont-Lezica, Plant Physiology, Biochemistry, and Biotechnology Emeritus Professor at the Universite de Toulouse, passed away on September 26, 2011 after losing his fight with cancer. He had studied agronomy at the Universidad Nacional de Cuyo, in Argentina, and in 1978 earned a Ph. D. at the Universite de Gembloux, Belgium. Rafael began his scientific career as a Research Assistant in Plant Physiology at the Universidad Nacional de Cuyo (1966–1971), where his work was centered on plant morphogenesis and secondary metabolism. He then became Assistant Professor in Biology at the Fundacion Bariloche, in Argentina (1972–1977), where he developed an interest in sugar metabolism, particularly in the synthesis of cellulose precursors and protein glycosylation. He obtained a Humboldt Stiftung Fellowship in Dr. Heinrich Kaus’ lab in Kaiserslautern, West Germany (1978–1979) and from 1979 to 1988 he was a Professor at the Universidad Nacional de Mar del Plata, in Argentina, where he co-founded, with Dr. Horacio Pontis, the Instituto de Investigaciones Biologicas. His main research topics there were on lectin biosynthesis and protein glycosylation. In the course of his first collaboration with Joe Varner, from Washington University in St. Louis, MO, USA, he focused his attention on the study of the plant cell wall. As a consequence he was invited as a visiting professor at Washington University in St. Louis, MO, USA (1987–1991). A crucial result of this fruitful collaboration was the development of the tissue printing technique, which became an alternative to in situ hybridization and immunocytochemistry. In 1992, Rafael obtained a professorship at the Universite de Toulouse and set up his own research group. Following his retirement in 2009, he became Emeritus Professor at the Universite de Toulouse. As such he kept up an intense research activity, interacting with his former team, today called Cell Wall Proteins and Development, in reference to his mentorship. Rafael's main research interest was the identification of Arabidopsis thaliana RGD-containing or integrin-like proteins involved in cell wall/plasma membrane contacts. He also began to work on plant cell wall proteomics. He presented the first overview of cell wall proteins and identified proteins that had never been described before. His most recent contribution was the setting up of two international research programs, introducing Brachypodium distachyon as a model plant for biomass production in our lab: European Knowledge-Based Bio-Economy (KBBE) and Comite Francais d'evaluation de la Cooperation Universitaire et Scientifique avec le Bresil (COFECUB). All these developments led to new insights in plant cell wall biology. At the Universite de Toulouse, Rafael directed the Plant Science Master's Program (1999–2004) and the European Marie Curie Training Program SIGNAL (Plant Signaling and Biotechnology; 2001–2005). He was a member of numerous scientific groups and councils at the local, national, and international levels [the Fundacion Bariloche, the Universidad Nacional de Mar del Plata, the Universite de Toulouse, the Ecole Doctorale de Biologie in Toulouse, the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) in Argentina, among others]. Although he developed his career in many different universities and countries, he stayed in touch with the colleagues with whom he had worked. Rafael was an appreciated teacher, always encouraging graduate students, postdocs, and young colleagues in the lab. His sound judgment and numerous personal qualities were appreciated and called upon by his colleagues and collaborators. As a researcher and a human being, he was enthusiastic and creative, communicating his drive and love of life to those around him.

The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton

Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5's ability to distinguish different forms of cell surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding protein, as intracellular factors able to alter TEM8 cell surface structure. Overexpression of either of these proteins in cells converted TEM8 from an SB5-exposed to an SB5-masked form and protected cells from SB5-saporin immunotoxins. Because the predominant form of TEM8 on the cell surface is not recognized by SB5, we also developed a new monoclonal antibody, called AF334, which is able to recognize both the SB5-exposed and the SB5-masked forms of TEM8. AF334-saporin selectively killed TEM8-positive cells independent of TEM8 cell surface structure. These studies reveal that TEM8 exists in different forms at the cell surface, a structure dependent on interactions with components of the actin cytoskeleton, and should aid in the rational design of the most effective diagnostic and therapeutic anti-TEM8 monoclonal antibodies.

Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin beta1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall-plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall-plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays.

β-Propeller Crystal Structure of Psathyrella velutina Lectin: An Integrin-like Fungal Protein Interacting with Monosaccharides and Calcium

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

Recombinant Mini-β3 Integrin Fragments for the Detection of HPA-1 Antibodies.

The αIIbβ3 integrin (GPIIbIIIa, CD61) is the platelet receptor for fibrinogen, fibronectin and vitronectin. A single nucleotide polymorphism (dbSNP Id= rs5918) in the ITGB3 gene defines the Human Platelet Antigen (HPA)-1 system encoding either a Leucine (1a) or Proline (1b) at position 33. HPA-1a immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post transfusion purpura (PTP) and platelet refractoriness. Currently, the detection of HPA-1 antibodies is based on a cumbersome sandwich ELISA requiring HPA genotyped platelets as source of antigen. We set out to express soluble calmodulin (CaM) tagged β3 fragments suitable for HPA-1 antibody detection. The design of fragments was informed by domain structural information, analysis of homology between β3 and integrin-like monomeric proteins and the evolutionary β3 domain phylogeny. Combined with computer modelling, this was the basis of the successful expression in Drosophila S2 cells of eight β3 fragments with either Leu33 or Pro33 (ΔSDL, ΔβA, PSI-Hybrid and PSI alone, both alleloforms for each of the four). Murine monoclonal antibodies Y2/51, SZ21 and CRC54 reacted with the β3 fragments as expected with only CRC54 reacting with the PSI fragment. Two of 3 human HPA-1a specific phage antibodies (19–7, 23–15, both derived from a PTP patient) reacted with all 4 Leu33 fragments but CamTran007 derived from a NAIT patient only reacted with the ΔSDL-Leu33 and ΔβA-Leu33 fragments, defining an epitope split. By ELISA, reactivity of both potency (NIBSC-03/152) and sensitivity (NIBSC-93/710) standards were comparable to those seen by MAIPA. Twelve polyclonal samples from NAIT patients (5 with cerebral haemorrhage [CH], 5 with platelet counts <40x109/L but no CH and 2 with counts >40x109/L) of potency ranging from 5–193 au/ml were detected by ELISA with Leu33 forms of ΔSDL and ΔβA. In conclusion soluble mini-β3-Leu33 fragments have been successfully expressed and shown to react with all polyclonal anti-HPA-1a samples tested from NAIT patients. The CaM tagged β3 fragments are now being further validated with a repository of 150 HPA-1a antibody samples from a NAIT case series with well defined treatment and clinical outcome histories to establish the relation between HPA-1a antibody potency, epitope reactivity and clinical phenotype.

A β4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: Inhibition of tyrosine phosphorylation slows tip growth

We present immunocytochemical data that indicate the presence of, and a close association between β4 integrin-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall–membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. By analogy with the integrins and associated kinases of the metazoa we suggest that these proteins could contribute to the process of tip growth by providing a means of bidirectional signaling between the cell wall and the cytoplasm.

Integrin-like protein at the invaginated plasma membrane of epidermal cells in mature leaves of the marine angiosperm Zostera marina L.

By immunoblotting with anti-human integrin beta polyclonal antibodies (beta1, beta3 or beta5), a single distinct band of about 60 kDa was detected in total protein extracts from mature leaves of the seagrass Zostera marina L., but no band was detected in total protein extracts from immature seagrass leaves, freshwater grass leaves or Arabidopsis thaliana (L.) Heynh. leaves. This integrin-like protein was detected by indirect immunofluorescence microscopy on the surface of non-spherical protoplasts of epidermal cells isolated from mature seagrass leaves using an anti-integrin beta3 polyclonal antibody. Electron-microscopic analyses with the same antibody indicated that this integrin-like protein was localized specifically in the invaginated plasma membrane of epidermal cells in mature seagrass leaves. Therefore, this integrin-like protein of about 60 kDa may be involved in the developmentally regulated invagination of the plasma membrane in epidermal cells of the seagrass Z. marina.

The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity.

Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin.

Differences in Candida albicans adhesion to intact and denatured type I collagen in vitro

An inhibition assay of Candida albicans adhesion to gelatin-immobilized membranes was compared with that to intact type I collagen-immobilized membranes using an arginine-glycine-aspartic acid (RGD) containing peptide. As compared with a protein-free membrane, gelatin and collagen significantly enhanced the adherence of C. albicans. The adhesion of the yeast to gelatin was significantly inhibited by the RGD peptides, but not by arginine-glycine-glutamic acid (RGE) peptides. In contrast, attachment to collagen was not inhibited by RGD peptides. These results suggest that the RGD sequence of gelatin and the integrin-like proteins of yeasts may be involved in adherence.