Loss of the Tumor Suppressor Pten Promotes Proliferation of Drosophila melanogaster Cells In Vitro and Gives Rise to Continuous Cell Lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (RasV12) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing RasV12. Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines

Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype

Averting biodiversity collapse in tropical forest protected areas

The rapid disruption of tropical forests probably imperils global biodiversity more than any other contemporary phenomenon¹⁻³. With deforestation advancing quickly, protected areas are increasingly becoming final refuges for threatened species and natural ecosystem processes. However, many protected areas in the tropics are themselves vulnerable to human encroachment and other environmental stresses⁴⁻⁹. As pressures mount, it is vital to know whether existing reserves can sustain their biodiversity. A critical constraint in addressing this question has been that data describing a broad array of biodiversity groups have been unavailable for a sufficiently large and representative sample of reserves. Here we present a uniquely comprehensive data set on changes over the past 20 to 30 years in 31 functional groups of species and 21 potential drivers of environmental change, for 60 protected areas stratified across the world’s major tropical regions. Our analysis reveals great variation in reserve ‘health’: about half of all reserves have been effective or performed passably, but the rest are experiencing an erosion of biodiversity that is often alarmingly widespread taxonomically and functionally. Habitat disruption, hunting and forest-product exploitation were the strongest predictors of declining reserve health. Crucially, environmental changes immediately outside reserves seemed nearly as important as those inside in determining their ecological fate, with changes inside reserves strongly mirroring those occurring around them. These findings suggest that tropical protected areas are often intimately linked ecologically to their surrounding habitats, and that a failure to stem broad-scale loss and degradation of such habitats could sharply increase the likelihood of serious biodiversity declines.Keywords: Ecology, Environmental scienc

<i>Pten</i> mutant primary cultures mirror the development of primary cultures expressing oncogenic <i>Ras^V12</i>.

<p>(A–C) Wild type. (D–F) <i>Ras^V12 (Act5C-Gal4; UAS-Ras^V12).</i> (G–I) <i>Pten¹¹⁷</i>. (J–L) <i>Pten¹¹⁷; Ras^V12 (Pten¹¹⁷; Act5C-Gal4/Pten¹¹⁷; UAS-Ras^V12).</i> After five days, primary cultures of all genotypes (A, D, G, J) had differentiated cell types including fat body (open arrowheads) and muscle (arrowheads). After ten days, wild-type cultures (B) had only the same differentiated cell types, whereas, cultures of the other genotypes (E, H, K) had patches of spindle-shaped cells. After 20 days, wild-type cultures (C) had only the same differentiated cell types, whereas, cultures of the other genotypes (F, I, L) were densely populated with spindle-shaped cells. (M and N) Western-blot analysis of primary culture extracts with cells of the indicated genotypes. (M) The Akt pathway is activated (pAkt) above control (wild type) levels in cultures with <i>Ras^V12</i> expressing cells <i>(Ras^V12), Pten</i> mutant cells (<i>Pten¹¹⁷</i>) and <i>Pten</i> mutant cells expressing <i>Ras^V12 (Pten¹¹⁷; Ras^V12).</i> The Erk pathway is activated (dpErk) above control (wild type) levels in cultures with <i>Ras^V12</i> expressing cells <i>(Ras^V12)</i> and <i>Pten</i> mutant cells expressing <i>Ras^V12 (Pten¹¹⁷; Ras^V12).</i> Total Akt and Erk, as detected by α-Akt and α-Erk were used as loading controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Gabay1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Kockel1" target="_blank">[41]</a>. Akt is detected as two bands (Cell Signaling Technology) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Willecke1" target="_blank">[31]</a>. For unknown reasons the lower band is more prominent in the <i>Pten</i> mutant cultures.</p

Generation and characteristics of <i>Pten</i> cell lines in reference to other cell lines.

<p>*surface area = 3.8 cm².</p><p>ANOVA and a Tukey-Kramer multiple comparison test showed that Pten and Pten; Ras lines had a significantly lower confluent density than Ras lines (P<0.01) (F value = 31.052 = MStreatment/MSresidual).</p

Morphological and molecular comparison of <i>Pten¹¹⁷</i> cell lines with S2, <i>Ras^V12</i> and <i>Pten¹¹⁷; Ras^V12</i> cell lines.

<p>(A–H) Cells are shown after 6 days of growth from the same starting cell number. (A) S2. Cells show the typical round morphology and are loosely attached to the surface. (B–D) <i>Pten¹¹⁷</i>. Pten 1 and 1A have round and spindle-shaped cells. Cells in the Pten 1 line are more loosely attached. Cells in line Pten X are primarily spindle shaped. (E–F) <i>Ras^V12</i>. Ras 7 and 3 have spindle shaped cells that form dense attached clusters. (G–H) <i>Pten¹¹⁷; Ras^V12.</i> Pten; Ras 8 and 9 have spindle shaped cells that form small clusters. (I) Western-blot analysis of cell extracts from the indicated lines. The Akt pathway is activated (pAkt) above control levels in all lines with <i>Pten</i> mutations. The Ras 7 line also shows elevated activation. The Erk pathway is activated (dpErk) in most lines with very low levels in S2 cells and undetectable levels in line Pten 1. Total Akt and Erk, as detected by α-Akt and α-Erk were used as loading controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Gabay1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Kockel1" target="_blank">[41]</a>. Akt is detected as two bands (Cell Signaling Technology) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417-Willecke1" target="_blank">[31]</a>.</p

Tumor suppressors tested in in vitro assays.

<p>*The allele used in this study is indicated. All alleles used were loss-of-function and when possible null alleles. The alleles and their sources are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#s3" target="_blank">methods</a> section.</p><p>**GO classification adapted from Panther (<a href="http://www.pantherdb.org/" target="_blank">http://www.pantherdb.org/</a>).</p

Time of appearance of proliferating cell patches in wild-type and tumor-suppressor mutant primary cultures.

<p>(A) Cross to generate embryos for tumor suppressor primary cultures. Tumor suppressor <i>(ts)</i> mutants were maintained in stocks with a marked balancer chromosome that expresses a <i>GFP</i> transgene <i>(Bal, GFP)</i>. One quarter of the progeny embryos are the desired class (boxed genotype). Primary cultures were established from mixed embryos and cells homozygous for the <i>ts</i> allele could be recognized because they are GFP negative. (B) The average day, and the range of days at which proliferating cells appear is shown. The number of cultures for each genotype is given (n). Wild-type control and <i>Act-Gal4; UAS-Ras^V12</i> positive control (blue); neoplastic mutants (red); hyperplastic mutants (green). The appearance of proliferating cells in <i>Pten</i> and <i>Ras^V12</i> cultures was significantly earlier than in wild-type control cultures (P<0.001). The appearance of proliferating cells in <i>wts</i> and <i>hippo</i> cultures was also significantly earlier than in wild-type control cultures (P<0.05). There were no significant differences between wild-type control cultures and any of the neoplastic mutants. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031417#pone.0031417.s001" target="_blank">Figure S1</a>. (C–F) Examples of cultures showing proliferating patches that first appeared on average at about 33–37 days in wild type (C) and <i>scribble</i> (D), 21 days in <i>hippo</i> (E) and 11 days in <i>Pten</i> (F). Differentiated cell types such as muscle (arrowhead) and fat (open arrowhead) are present in all genotypes. Insets in D–F show a GFP image demonstrating that the indicated patches of proliferating cells (arrows) were negative for GFP and therefore of the mutant genotype.</p

LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis