Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Specific tracheal migration is mediated by complementary expression of cell surface proteins.

Migration of the Drosophila tracheal cells relies on cues provided by nearby cells; however, little is known about how these signals specify a migratory path. Here we investigate the role of cell surface proteins in the definition of such a pathway. We have found that the PS1 integrin is required in the tracheal cells of the visceral branch, whereas the PS2 integrin is required in the visceral mesoderm; both integrins are necessary for the spreading of the visceral branch over its substratum. This is the first identification of a cell surface molecule with expression restricted to a subset of tracheal cells that all migrate in a given direction. We have also found that expression of PS1 in the visceral branch is regulated by the genes that direct tracheal cell migration, showing that integrin expression is part of the cell-fate program that they specify. These results support a model in which signal transduction determines the tracheal migratory pathways by regulating the expression of cell surface proteins, which in turn interact with surface molecules on the surrounding cell population.

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

The localized assembly of extracellular matrix integrin ligands requires cell-cell contact.

The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. We have examined this by testing whether the PS2 integrin can recruit tiggrin to ectopic locations within the Drosophila embryo. We found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for tiggrin, can recruit tiggrin to new cellular contexts. Next, we genetically manipulated the fate of the muscles and the epidermal muscle attachment cells, which demonstrated that muscles have the primary role in recruiting tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus we propose that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly.

An integrin chicken and egg problem: which comes first, the extracellular matrix or the cytoskeleton?

Integrins have the ability to organise macromolecular structures both inside and outside the cell. Analysis of integrin function in the developing embryos of worms and flies suggests that, although the extracellular matrix directs integrins to organise intracellular proteins, the cytoskeleton may have the first word.

Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system.

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.

Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions.

Cell migration during embryogenesis involves two populations of cells: the migrating cells and the underlying cells that provide the substratum for migration. The formation of the Drosophila larval midgut involves the migration of the primordial midgut cells along a visceral mesoderm substratum. We show that integrin adhesion receptors are required in both populations of cells for normal rates of migration. In the absence of the PS integrins, the visceral mesoderm is disorganised, the primordial midgut cells do not display their normal motile appearance and their migration is delayed by 2 hours. Removing PS integrin function from the visceral mesoderm alone results in visceral mesoderm disorganization, but only causes a modest delay in migration and does not affect the appearance of the migrating cells. Removing PS integrin function from the migrating cells causes as severe a delay in migration as the complete loss of PS integrin function. The functions of PS1 and PS2 are specific in the two tissues, endoderm and mesoderm, since they cannot substitute for each other. In addition there is a partial redundancy in the function of the two PS integrins expressed in the endoderm, PS1 (alphaPS1betaPS) and PS3 (alphaPS3betaPS), since loss of just one alpha subunit in the midgut results in either a modest delay (alphaPS1) or no effect (alphaPS3). We have also examined the roles of small GTPases in promoting migration of the primordial midgut cells. We find that dominant negative (N17) versions of Rac and Cdc42 cause a very similar defect in migration as loss of integrins, while those of Rho and Ras have no effect. Thus integrins are involved in mediating migration by creating an optimal substratum for adhesion, adhering to that substratum and possibly by activating Rac and Cdc42.

Reproducibility of guidelines. A comparison of the NCCN and ASCO lung cancer guidelines.

Clinical practice guidelines are reproducible if two groups of experts, presented with the same evidence and methods, derive similar recommendations. Reproducibility is an essential attribute in justifying the use of clinical practice guidelines to guide and monitor physicians' practices. This article will compare guidelines for the management of advanced non-small-cell lung cancer developed by the National Comprehensive Cancer Network (NCCN) with guidelines derived by the American Society of Clinical Oncology (ASCO). This comparison aims to determine whether the two guidelines are similar or whether there are major differences that pose significant problems for clinicians attempting to manage patients according to one oncology standard.

Uncoupling integrin adhesion and signaling: the betaPS cytoplasmic domain is sufficient to regulate gene expression in the Drosophila embryo.

Integrin cell surface receptors are ideally suited to coordinate cellular differentiation and tissue assembly during embryogenesis, as they can mediate both signaling and adhesion. We show that integrins regulate gene expression in the intact developing embryo by identifying two genes that require integrin function for their normal expression in Drosophila midgut endodermal cells. We determined the relative roles of integrin adhesion versus signaling in the regulation of these integrin target genes. We find that integrin-mediated adhesion is not required between the endodermal cells and the surrounding visceral mesoderm for integrin target gene expression. In addition, a chimeric protein that lacks integrin-adhesive function, but maintains the ability to signal, can substitute for the endogenous integrin and regulate integrin target genes. This chimera consists of an oligomeric extracellular domain fused to the integrin betaPS subunit cytoplasmic domain; a control monomeric extracellular domain fusion does not alter integrin target gene expression. Therefore, oligomerization of the 47-amino-acid betaPS intracellular domain is sufficient to initiate a signaling pathway that regulates gene expression in the developing embryo.

kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin.

Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.

Guidelines and disease management.

The four NCCN Guidelines that follow represent the initial deliberations of an institutionally diverse, multidisciplinary panel comprised of internationally recognized experts. These experts have attempted to address the broad range of clinical decisions that oncologists face as they attempt to manage a patient with a particular tumor. The goal of the NCCN guidelines program is to publish comprehensive pathways that will serve as a foundation for all cancer care providers to develop superior disease management programs.

Modulation of integrin activity is vital for morphogenesis.

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the alpha subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (alphaPS2betaPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type alphaPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the alphaPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the alphaPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin alphaIIbbeta3.

Absence of PS integrins or laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos.

We have examined the role of integrins in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). To this end we have analyzed muscle attachments and neuromuscular junctions ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions (HAJs) to extracellular matrix, but not for their intracellular link to the cytoskeleton. The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. It is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component laminin A, while adhesion mediated by PS2 integrin does not. At neuromuscular junctions the formation of functional synapses occurs normally in embryos lacking PS integrins and/or laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly. We suggest that neuromuscular contact in part requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of laminin A.

Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions.

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. In order to investigate whether PS2 integrin is required at these additional sites and/or has functions besides mediating adhesion, a comprehensive genetic analysis of inflated, the gene that encodes alphaPS2, was performed. We isolated 35 new inflated alleles, and obtained 10 alleles from our colleagues. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signalling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing.

The NCCN guideline program--1998.

In 1995, the National Comprehensive Cancer Network (NCCN) embarked on an ambitious project to develop comprehensive diagnostic and treatment algorithms for the broad spectrum of oncologic diseases and support modalities. To date, 28 NCCN guideline panels have developed guidelines covering an estimated 93% of tumors, and an additional 9 panels have guidelines under development. This article reviews the NCCN guideline infrastructure and development process. The process relies on the expertise of the 17 NCCN member institutions and follows a formal procedure for guideline development to ensure that the recommendations offer the cancer patient the best chance for a successful outcome.

Specificity of PS integrin function during embryogenesis resides in the alpha subunit extracellular domain.

We tested the ability of different integrin alpha subunits to substitute for each other during embryonic development. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers where they are required for normal development. Using the GAL4 system, we are able to rescue fully the embryonic lethality of an alphaPS2 null mutation with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, due to partial rescue of both muscle and midgut phenotypes. Similarly we are able to rescue the embryonic/first instar larval lethality of an alphaPS1 null mutation gene with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. We conclude that the two alpha subunits are not equivalent and have distinct functions which reside in the extracellular domains.