Architectural and Functional Diversity of Polycomb Group Response Elements in Drosophila

Polycomb group response elements (PREs) play an essential role in gene regulation by the Polycomb group (PcG) repressor proteins in Drosophila. PREs are required for the recruitment and maintenance of repression by the PcG proteins. PREs are made up of binding sites for multiple DNA-binding proteins, but it is still unclear what combination(s) of binding sites is required for PRE activity. Here we compare the binding sites and activities of two closely linked yet separable PREs of the Drosophila engrailed (en) gene, PRE1 and PRE2. Both PRE1 and PRE2 contain binding sites for multiple PRE–DNA-binding proteins, but the number, arrangement, and spacing of the sites differs between the two PREs. These differences have functional consequences. Both PRE1 and PRE2 mediate pairing-sensitive silencing of mini-white, a functional assay for PcG repression; however, PRE1 requires two binding sites for Pleiohomeotic (Pho), whereas PRE2 requires only one Pho-binding site for this activity. Furthermore, for full pairing-sensitive silencing activity, PRE1 requires an AT-rich region not found in PRE2. These two PREs behave differently in a PRE embryonic and larval reporter construct inserted at an identical location in the genome. Our data illustrate the diversity of architecture and function of PREs.     

Evolutionarily Conserved Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A/B Proteins Functionally Interact with Human and Drosophila TAR DNA-binding Protein 43 (TDP-43)

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.     

The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.     

Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.     

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis,Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.     

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-to-cell movement (for review, see Benitez-Alfonso et al., 2010; Laliberté and Sanfaçon, 2010; Niehl and Heinlein, 2011; Ueki and Citovsky, 2011; Verchot, 2012). It is accepted that plant virus replication is associated with host membranes, and at some point, the viral genomic nucleic acid must be transferred from the site of replication in the cell to the plasmodesmata. This step could involve transport from a distant site within the cell, or alternatively, it may be that replication is coupled with transport at the entrance of the plasmodesmata (Tilsner et al., 2013). However, even with the latter model, there is ample evidence that the viral proteins necessary for replication or cell-to-cell movement utilize intracellular trafficking pathways within the cell to become positioned at the plasmodesma. These pathways may involve microfilaments, microtubules, or specific endomembranes that participate in macromolecular transport pathways, or combinations of these elements (Harries et al., 2010; Schoelz et al., 2011; Patarroyo et al., 2012; Peña and Heinlein, 2012; Tilsner and Oparka 2012; Liu and Nelson, 2013).The P6 protein of Cauliflower mosaic virus (CaMV) is one viral protein that had not been considered to play a role in viral movement until recently. P6 is the most abundant protein component of the amorphous, electron-dense inclusion bodies (IBs) present during virus infection (Odell and Howell, 1980; Shockey et al., 1980). Ectopic expression of P6 in Nicotiana benthamiana leaves resulted in the formation of inclusion-like bodies (I-LBs) that were capable of intracellular movement along actin microfilaments. Furthermore, treatment of Nicotiana edwardsonii leaves with latrunculin B abolished the formation of CaMV local lesions, suggesting that intact microfilaments are required for CaMV infection (Harries et al., 2009a). A subsequent paper showed that P6 physically interacts with Chloroplast Unusual Positioning1 (CHUP1), a plant protein localized to the chloroplast outer membrane that contributes to movement of chloroplasts on microfilaments in response to changes in light intensity (Oikawa et al., 2003, 2008; Angel et al., 2013). The implication was that P6 might hijack CHUP1 to facilitate movement of the P6 IBs on microfilaments. Silencing of CHUP1 in N. edwardsonii, a host for CaMV, slowed the rate of local lesion formation, suggesting that CHUP1 contributes to intracellular movement of CaMV (Angel et al., 2013).In addition to its role in intracellular trafficking, the P6 protein has been shown to have at least four other distinct functions in the viral infection cycle. P6-containing IBs induced during virus infection are likely virion factories, as they are the primary site for CaMV protein synthesis, genome replication, and assembly of virions (Hohn and Fütterer, 1997). Second, P6 interacts with host ribosomes to facilitate reinitiation of translation of genes on the polycistronic 35S viral RNA, a process called translational transactivation (Bonneville et al., 1989; Park et al., 2001; Ryabova et al., 2002). The translational transactivator region of P6 (Fig. 1) defines the essential sequences required for translational transactivation (DeTapia et al., 1993). Third, P6 is an important pathogenicity determinant. P6 functions as an avirulence determinant in some solanaceous and cruciferous species (Daubert et al., 1984; Schoelz et al., 1986; Hapiak et al., 2008) and is a chlorosis symptom determinant in susceptible hosts (Daubert et al., 1984; Baughman et al., 1988; Goldberg et al., 1991; Cecchini et al., 1997). Finally, P6 has the capacity to compromise host defenses, as it is a suppressor of RNA silencing and cell death (Love et al., 2007; Haas et al., 2008), and it modulates signaling by salicylic acid, jasmonic acid, ethylene, and auxin (Geri et al., 2004; Love et al., 2012; Laird et al., 2013). Domain D1 of P6 has been shown to be necessary but not sufficient for suppression of silencing and salicylic acid-mediated defenses (Laird et al., 2013).Open in a separate windowFigure 1.CaMV and host constructs used for confocal microscopy or coimmunoprecipitation (co-IP). A, Structure of CaMV P6 and Arabidopsis (Arabidopsis thaliana) Soybean Response to Cold (AtSRC2.2) proteins. The functions of P6 domains D1 to D4 tested for interaction with AtSRC2.2 are indicated by the shaded boxes. The Mini TAV is the minimal region for the translational transactivation function. The NLSa sequence corresponds to the nuclear localization signal of influenza virus. The NLS sequence corresponds to the nuclear localization signal of human ribosomal protein L22. B, Structure of P6 (Angel et al., 2013), AtSRC2.2, PDLP (Thomas et al., 2008), and CaMV MP fusions developed for confocal microscopy and/or co-IP. aa, Amino acid.Because P6-containing IBs are the site for virion accumulation and they are capable of movement, they may be responsible for delivering virions to the CaMV MP located at the plasmodesmata (for review, see Schoelz et al., 2011). The vast majority of CaMV virions accumulate in association with P6-containing IBs. Furthermore, P6 physically interacts with the CaMV capsid and MP, as well as the two proteins necessary for aphid transmission, P2 and P3 (Himmelbach et al., 1996; Ryabova et al., 2002; Hapiak et al., 2008; Lutz et al., 2012). Recent studies have indicated that P6 IBs serve as a reservoir for virions, in which the virions may be rapidly transferred to P2 electron-lucent IBs for acquisition by aphids (Bak et al., 2013). It stands to reason that P6 IBs may also serve as a reservoir for CaMV virions to be transferred to the CaMV MP in the plasmodesmata.CaMV virions move from cell to cell through plasmodesmata modified into tubules through the function of its MP (Perbal et al., 1993; Kasteel et al., 1996). However, studies have suggested that CaMV virions do not appear to directly interact with the MP. Instead, the MP interacts with the CaMV P3 protein (also known as the virion-associated protein [VAP]), which forms a trimeric structure that is anchored into the virions (Leclerc et al., 1998; Leclerc et al., 2001). Electron microscopy studies have indicated that MP and VAP colocalize with virions only at the entrance to or within the plasmodesmata, and it has been suggested that the VAP/virion complex travels to the plasmodesmata independently from the MP (Stavolone et al., 2005). Consequently, there is a need for a second CaMV protein such as P6 to fulfill the role of delivery of virions to the plasmodesmata (Schoelz et al., 2011).Additional studies have shown that the CaMV MP is incorporated into vesicles and is trafficked on the endomembrane system to reach the plasmodesma (Carluccio et al., 2014). These authors suggest that the CaMV MP is recycled in a vesicular transport pathway between plasmodesmata and early endosome compartments. The CaMV MP interacts with µA-Adaptin (Carluccio et al., 2014) and Movement Protein-Interacting7 (Huang et al., 2001), two proteins shown to have a role in vesicular trafficking. Once the MP arrives at plasmodesmata, it interacts with the Plasmodesmata-Localized Protein (PDLP) proteins, which comprise a family of eight proteins associated with plasmodesmata (Amari et al., 2010). In addition to its interaction with CaMV MP, PDLP1 interacts with the 2B protein of Grapevine fan leaf virus (GFLV) at the base of tubules formed by the 2B protein. Furthermore, an Arabidopsis transfer DNA (T-DNA) mutant line in which three PDLP genes had been knocked out (pdlp1-pdlp2-pdlp3) responded to GFLV and CaMV inoculation with a delayed infection (Amari et al., 2010). This has led to the suggestion that the PDLPs might act as receptors for the MPs of the tubule-forming viruses such as GFLV and CaMV (Amari et al., 2010, 2011).To better understand the function of the P6 protein during CaMV intracellular movement, we have utilized a yeast (Saccharomyces cerevisiae) two-hybrid assay to identify host proteins that interact with CaMV P6. We show that P6 physically interacts with a C2-calcium-dependent protein (designated AtSRC2.2). AtSRC2.2 is a membrane-bound protein that is capable of forming punctate spots associated with plasmodesmata. The localization of AtSRC2.2 with plasmodesmata led to an analysis of interactions between P6 I-LBs, AtSRC2.2, PDLP1, and the CaMV MP and also revealed that a portion of P6 I-LBs are found adjacent to plasmodesmata. These results provide further evidence for a model in which P6 IBs are capable of delivery of virions to plasmodesmata for their transit to other host cells.     

Identification of Immune System and Response Genes, and Novel Mutations Causing Melanotic Tumor Formation in Drosophila Melanogaster

We are using Drosophila as a model system for analysis of immunity and tumor formation and have conducted two types of screens using enhancer detector strains to find genes related to these processes; genes expressed in the immune system (type A; hemocytes, lymph glands and fat body) and genes increased in expression by bacterial infection (type B). For type A, tissue-specific reporter gene activity was determined. For type B, a variation of enhancer detection was devised in which β-galactosidase is assayed spectrophotometrically with and without bacterial infection. Because of immune system involvement in melanotic tumor formation, a third type was hypothesized to be found among types A and B: genes that, when mutated, have a melanotic tumor phenotype. Enhancer detector strains (2800) were screened for type A, 900 for B, and 11 retained for further analysis. Complementation tests, cytological mapping, P-element mobilization, and determination of lethal phase and mutant phenotype have identified six novel genes, Dorothy, wizard, toto, viking, Thor and dappled, and one previously identified gene, Collagen IV. All are associated with reporter gene expression in at least one immune system tissue. Thor has increased expression upon infection. Mutations of wizard and dappled have a melanotic tumor phenotype.     

A Cluster of Cuticle Protein Genes of Drosophila Melanogaster at 65a: Sequence, Structure and Evolution

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.     

Comparative Genomics and Proteomics of Drosophila, Brenner's Nematode, and Arabidopsis: Identification of Functionally Similar Genes and Proteins of Meiotic Chromosome Synapsis

The published principles of computer analysis of genomes and protein sets in taxonomically distant eukaryotes are expounded. The authors developed a search strategy to identify in genomes of such organisms genes and proteins nonhomologous in primary structure but having similar functions in cells dividing by meiosis. This strategy based on the combined principles of genomics, proteomics, and morphometric analysis of subcellular structures was applied to a computer search for genes encoding the proteins of synaptonemal complexes in genomes of Drosophila melanogaster, the nematode Caenorhabditis elegans, and the plant Arabidopsis thaliana. These proteins proved to be functionally similar to their counterparts in yeast Saccharomyces cerevisiae (protein Zip1p) and mammals (protein SCP1).     

Three Heat Shock Protein Genes from Bactrocera (Tetradacus) minax Enderlein: Gene Cloning,Characterization, and Association with Diapause

Bactrocera (Tetradacus) minax Enderlein is a major pest to wild and cultivated species of citrus. Bactrocera minax produces one generation per year with a long pupal diapause period of over 6 months, which hinders efforts to obtain vast numbers of insects under standard room conditions. Determining the mechanisms of diapause is significantly important for obtaining large quantities of these insects. To characterize the heat shock protein (Hsp) genes of B. minax and to unravel their potential contribution to diapause, we performed 3′ and 5′ RACE to isolate the complementary DNA (cDNA) sequences, bioinformatics to examine the phylogenetic relationships, and real-time quantitative PCR to detect the expression patterns of three Hsp genes during various developmental stages. These results represent the first characterization of the three Hsp genes of B. minax; the open reading frames of Bmhsp23, Bmhsp70, and Bmhsp90 were 510, 1,911, and 1,089 bp, encoding 170, 636, and 363 amino acids, respectively. BmHsp70 and BmHsp90 displayed high identity to previously identified Hsp70 and Hsp90 genes, respectively. BmHsp23 displayed varying similarity, from 28 to 83%, to previously identified small Hsps. Bmhsp23 messenger RNA (mRNA) expression was found to be upregulated during diapause initiation, maintenance, and termination. Bmhsp70 mRNA expression peaked during diapause initiation. Bmhsp90 mRNA expression remained at a relatively low level during deep diapause. Our present results suggest that Bmhsp70 might play an important role in diapause initiation, while Bmhsp23 in diapause initiation and maintenance and Bmhsp90 in diapause regulation. These results improve our understanding of the mechanism of diapause in B. minax at the molecular level.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.     

FUS/TLS forms cytoplasmic aggregates,inhibits cell growth and interacts with TDP-43 in a yeast model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the premature loss of motor neurons. While the underlying cellular mechanisms of neuron degeneration are unknown, the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease. Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients. It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates. Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates. We find that FUS and TDP-43 have a high propensity for co-aggregation, unlike the aggregation patterns of several other aggregation-prone proteins. Moreover, the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins, suggesting they are not composed of amyloid.     

Increases in mRNA and Protein Levels of the Genes for the Actin-Binding Proteins Profilin,Fascin, and Ezrin Promote Metastasis in Non-Small Cell Lung Cancer

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC.