29 research outputs found
PTEN redundancy: overexpressing lpten, a homolog of Dictyostelium discoideum ptenA, the ortholog of human PTEN, rescues all behavioral defects of the mutant ptenA-.
Mutations in the tumor suppressor gene PTEN are associated with a significant proportion of human cancers. Because the human genome also contains several homologs of PTEN, we considered the hypothesis that if a homolog, functionally redundant with PTEN, can be overexpressed, it may rescue the defects of a PTEN mutant. We have performed an initial test of this hypothesis in the model system Dictyostelium discoideum, which contains an ortholog of human PTEN, ptenA. Deletion of ptenA results in defects in motility, chemotaxis, aggregation and multicellular morphogenesis. D. discoideum also contains lpten, a newly discovered homolog of ptenA. Overexpressing lpten completely rescues all developmental and behavioral defects of the D. discoideum mutant ptenA-. This hypothesis must now be tested in human cells
<i>lpten<sup>ā</sup></i> cells translocating in buffer in the absence of chemoattractant exhibit defects in velocity, turning and the suppression of lateral pseudopod formation.
<p>Cells were analyzed in a perfusion chamber through which buffer without attractant was pumped. A. 2D motility parameters of Ax2, <i>lpten<sup>ā</sup></i> and <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> cells assessed with 2D-DIAS software. B, C, D. 2D-DIAS reconstructions of cell perimeters to generate tracks. Arrows denote net direction, and the blue-filled perimeters represent the last cell positions in the tracks. E, F. 3D-DIAS reconstructions at 0Ā° (top view) and 90Ā° (side view) of representative Ax2 and <i>lpten<sup>ā</sup></i> cells, respectively, denoting pseudopods (red). Note that the multiple lateral pseudopods formed by <i>lpten<sup>ā</sup></i> cells, were primarily off the substrate. a, anterior end of cell; p, posterior end of cell; lps, lateral pseudopod. G. 2D analysis of lateral pseudopod formation. Inst. vel., instantaneous velocity; No. turns per 10 min., number of turns per 10 minutes; Percent mot. cells, percent motile cells. Parameters are presented as the means Ā± standard deviations. T-test was used to determine p values. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p
Overexpressing <i>lpten<sup>ā</sup></i> in the <i>ptenA<sup>ā</sup></i> mutant.
<p>A. The transformation vector used to generate strains <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i>, in which <i>lpten</i> is under the regulation of the <i>actin 15</i> (<i>act15</i>) promoter, fused in frame at the 3ā² end to the red fluorescent protein gene (<i>rfp</i>) and terminating with a 3ā² <i>actin 8</i> gene sequence. The positions of the primers P8 and P9, for generating the <i>lpten-rfp</i> cDNA, are denoted. Insert shows verification of the <i>lpten-rfp</i> cDNA by PCR. B. <i>lpten</i> is expressed in <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> cells at levels more than 10 times that in the parent <i>ptenA<sup>ā</sup></i> mutant. The positions of the primers (P1, P2) for RT-PCR of the 300 bp <i>lpten</i> fragment (F) are denoted. In the insert to the right of panel B, RT-PCR products of chemotactically responsive <i>ptenA<sup>ā</sup></i> and <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> cells reveals overexpression of <i>lpten</i> in the latter. Densitometry measurements revealed>10 fold overexpression. C. Fruiting body formation in Ax2 cultures. D. The absence of fruiting body formation in <i>ptenA<sup>ā</sup></i> cultures. E. Fruiting body formation in <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> cultures. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s001" target="_blank">Table S1</a> for description of primers.</p
Cultural characteristics and virulence of strains of Fusobacterium necrophorum isolated from the feet of cattle and sheep
<i>lpten<sup>ā</sup></i> cells undergo normal chemotaxis in the low cAMP concentration gradient generated in the estimated for that of the natural wave.
<p>The gradient was generated in BSS buffer, in which K<sup>+</sup> and Na<sup>+</sup> are the facilitating cations. <i>lpten<sup>ā</sup></i> cells, however, are still defective in suppressing lateral pseudopod formation. A. 2D motility and chemotaxis parameters, assessed by 2D-DIAS software of Ax2, <i>lpten<sup>ā</sup></i> and <i>lpten<sup>ā</sup>/lpten<sup>oe</sup></i> cells undergoing chemotaxis in a low cAMP concnetration gradient. B, C, D. 2D-DIAS-reconstructed perimeter tracks of representative cells. The large arrows at panel bottoms denote the net direction of the increasing cAMP gradient. āSinkā, trough with buffer alone; āSourceā, trough with buffer plus 1 ĀµM cAMP. E. 2D analysis of lateral pseudopod formation. Direct. Persist, directional persistence; chem. index, Chemotactic Index (CI); Percent pos. chem., percent cells with a positive CI. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g002" target="_blank">Figure 2</a> for additional definitions and details. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p
Overexpression of <i>lpten</i> rescues the behavioral defects exhibited by homogeneous populations of <i>ptenA<sup>ā</sup></i> cells undergoing chemotaxis in natural aggregation territories in submerged cultures on glass.
<p>A, B, C. The centroid tracks of four neighboring cells representative of the general behavior of Ax2, <i>ptenA<sup>ā</sup></i> and <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> populations, respectively, are presented in relation to the aggregation centers of Ax2 and <i>ptenA<sup>ā</sup>/lpten<sup>oe</sup></i> cells, and the interpreted aggregation center of <i>ptenA<sup>ā</sup></i>, deduced retrospectively by the direction of net translocation of groups of cells, in the upper half of each panel. The first (1) and last (150) centered in the centroid tracks are noted. In lower half of each panel, the velocity plots are presented for two respective cells. For normal cells, the peaks of velocity have been shown to correlate with the front of each relayed natural wave.</p
Chemotactic behavior of <i>ptenA<sup>ā</sup></i> cells pulsed with cAMP to achieve chemotactic competence: a comparison of four different studies involving either ālow cAMP concentrationsā in the estimated range for natural cAMP waves or āhigh cAMP concentration gradientsā, at concentrations 10 times that of natural cAMP waves.
<p>a. Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Iijima1" target="_blank">[29]</a>: Iijima and Devreotes, 2002; reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Hoeller1" target="_blank">[32]</a>: Hoeller and Kay, 2007; reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Wessels1" target="_blank">[30]</a>, Wessels et al., 2007.</p><p>b. Complete composition of buffers used in chemotaxis assay. DB buffer: 5 mM Na<sub>2</sub>HPO<sub>4</sub>, 5 mM NaH<sub>2</sub>PO<sub>4,</sub> 2 mM MgSO<sub>4</sub>, 0.2 mM CaCl<sub>2</sub> (15 mM Na<sup>+</sup>, 0.2 mM Ca<sup>2+</sup>). KK<sub>2</sub> buffer: 3.9 mM K<sub>2</sub>HPO<sub>4</sub>, 16.5 mM KH<sub>2</sub>PO<sub>4</sub>, 2 mM Mg SO<sub>4</sub>, 0.1 mM CaCl<sub>2</sub> (24.3 mM K<sup>+</sup>, 0.1 mM CA<sup>2+</sup>, 0.1 mM Ca<sup>2+</sup>). BSS buffer: 20 mM KH<sub>2</sub>PO<sub>4</sub>, 5 mM NA<sub>2</sub>HPO<sub>4</sub>, 20 mM KCL, 2.5 mM MgCl<sub>2</sub> (45 mM K<sup>+</sup>/Na<sup>+</sup>, 0 mM Ca<sup>2+</sup><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Lusche3" target="_blank">[77]</a>.</p><p>c. When the source of the cAMP gradient was 1 ĀµM, it generated a ālow cAMP concentration gradientā, in the concentration range estimated for the natural wave <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Tomchik1" target="_blank">[44]</a>, and when it was 10 ĀµM, it generated a high cAMP concentration gradient 10 times that estimated for the natural.</p><p>Chemotactic behavior of <i>ptenA<sup>ā</sup></i> cells pulsed with cAMP to achieve chemotactic competence: a comparison of four different studies involving either ālow cAMP concentrationsā in the estimated range for natural cAMP waves or āhigh cAMP concentration gradientsā, at concentrations 10 times that of natural cAMP waves.</p