Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D.discoideum

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum. However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25–50 nM, and dinitrophenol (DNP), at 2.5–5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1–2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity

Differentiation-Inducing Factor-1 and -2 Function also as Modulators for Dictyostelium Chemotaxis

BackgroundIn the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions.Methodology/Principal FindingsTo further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP]i). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP]i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part.Conclusions/SignificanceOur findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity

Dictyostelium: An Important Source of Structural and Functional Diversity in Drug Discovery

The cellular slime mold Dictyostelium discoideum is an excellent model organism for the study of cell and developmental biology because of its simple life cycle and ease of use. Recent findings suggest that Dictyostelium and possibly other genera of cellular slime molds, are potential sources of novel lead compounds for pharmacological and medical research. In this review, we present supporting evidence that cellular slime molds are an untapped source of lead compounds by examining the discovery and functions of polyketide differentiation-inducing factor-1, a compound that was originally isolated as an inducer of stalk-cell differentiation in D. discoideum and, together with its derivatives, is now a promising lead compound for drug discovery in several areas. We also review other novel compounds, including secondary metabolites, that have been isolated from cellular slime molds

Effects of DIF-1 and DIF-2 on chemotaxis and intracellular cGMP in Ax2, <i>regA^-</i>, and <i>gbpB^-</i> cells.

<p>(A, B) Cells starved for 6 h were spotted on PB agar containing 3 mM caffeine (Control) plus 100 nM (A) or 10 nM (B) of DIF-1 or DIF-2 and assayed for chemotaxis toward the indicated doses of cAMP (top). Starved cells in shake-culture were stimulated with 0.3 nM cAMP (final concentration) in the presence of 3 mM caffeine (Control) plus 100 nM (A) or 10 nM (B) of DIF-1 or DIF-2, and aliquots of the cells were collected for assay of cGMP contents (bottom). Data are the mean and s.d. (bars) of three independent experiments (n = 3). *<i>P</i><0.05, as compared with Control.</p

Derivatives of Differentiation-Inducing Factor 1 Differentially Control Chemotaxis and Stalk Cell Differentiation in <i>Dictyostelium discoideum</i>

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation–inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum

Effects of DIF-1 and DIF-2 on chemotaxis of various mutants.

<p>Various mutants starved for 6 h were spotted on PB agar containing 3 mM caffeine (Control) plus 100 nM DIF-1 or DIF-2 and assayed for chemotaxis toward the indicated doses of cAMP. Data are the mean and s.d. (bars) of three independent experiments (n = 3). *<i>P</i><0.05, as compared with Control. <i>pi3k1</i> and <i>pi3k2</i>: genes of phosphoinositide-3 kinase. <i>pten</i>: phasphotase and tensin homolog gene. <i>plaA</i>: phospholipase A2 gene. <i>gca</i> and <i>sgc</i>: genes of guanylyl cyclase A and soluble guanylyl cyclase. <i>gbpA, gbpB</i>, and <i>gbpD</i>: genes of cGMP-binding protein A, B, and D, respectively. Note that chemotaxis toward low concentrations of cAMP was impaired in <i>pi3k1^-/2^-, pten^-, plaA^-</i>, and <i>gca^-/sgc</i>^-, in which the chemotaxis-modulating effects of DIFs were not observed, and that DIFs showed essentially the same effects in <i>gbpA^-/B^-</i> as seen in <i>gbpB^-</i> cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006658#pone-0006658-g006" target="_blank">Fig. 6A</a>).</p

Effects of DIF-1 and DIF-2 on chemotaxis in Ax2 and HM1030 (<i>dmtA^-</i>) cells.

<p>(A) Chemical structure of DIF-1: 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one and DIF-2: 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)pentan-1-one. (B) Ax2 and HM1030 cells were starved for 4–8 h (as indicated in parentheses) in shake-culture, and cell droplets were spotted on PB agar containing 3 mM caffeine (Control) plus 100 nM DIF-1 or DIF-2. Cells were assayed for chemotaxis toward the indicated doses of cAMP (10 cell droplets were examined for each cAMP concentration). Data are the mean and s.d. (bars) of three independent experiments (n = 3). *<i>P</i><0.05, as compared with Control.</p

Effects of DIF-1 and DIF-2 on chemotaxis in Dim mutants.

<p>(A) Starved (for 6 h) <i>dimA</i>^-, <i>dimB</i>^-, and <i>dimA</i>^-<i>/B</i>^- cells were spotted on PB agar containing 3 mM caffeine (Control) plus 100 nM DIF-1 or DIF-2 and assayed for chemotaxis toward the indicated doses of cAMP. Data are the mean and s.d. (bars) of three independent experiments (n = 3). (B) Starved <i>dimA</i>^-, <i>dimB</i>^-, and <i>dimA</i>^-<i>/B</i>^- cells were spotted on PB agar containing 3 mM caffeine plus the indicated concentrations of DIF-1 or DIF-2 and assayed for chemotaxis toward the doses of cAMP indicated above in square brackets. Data are the mean and s.d. (bars) of three independent experiments (n = 3). *<i>P</i><0.05, as compared with Control. (C) Expression levels of <i>regA</i> and <i>gbpB</i>. Cells were starved for 6 h, and RNAs collected from the cells were used for semi-quantitative RT-PCR to detect <i>regA, gbpB</i>, and <i>rnlA</i> (internal control).</p