BACKGROUND & AIMS:
: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.
背景与目标:
: 分化诱导因子-1 [1-(3,5-二氯-2,6-二羟基-4-甲氧基苯基) hexan-1-one (DIF-1)] 是细胞黏菌发育过程中细胞分化和趋化性的重要调节剂DIF-1下游的整个信号通路仍有待阐明。为了表征DIF-1及其潜在受体,我们合成了两种DIF-1的荧光衍生物,硼-二吡咯亚甲基 (BODIPY)-共轭DIF-1 (DIF-1-BODIPY) 和硝基苯并恶二唑 (NBD)-共轭DIF-1 (DIF-1-NBD),并研究了它们的生物学活性和细胞定位。在环腺苷单球 (cAMP) 存在下,DIF-1-BODIPY (5 µ m) 和DIF-1 (2 nM) 诱导DIF缺陷菌株HM44的茎细胞分化,而DIF-1-NBD (5 µm µ m) 在相同条件下几乎不诱导茎细胞分化。显微分析显示,生物活性衍生物DIF-1-BODIPY在分化后期被茎细胞掺入,并定位于线粒体。线粒体解偶联羰基氰化物间氯苯基腙 (CCCP),在cAMP. DIF-1-BODIPY (1-2 µ m) 和DIF-1 (10 nM) 以及CCCP和DNP的存在下,HM44在25-50 nM和2.5-5 µ m诱导茎秆细胞部分分化,在浅cAMP梯度中抑制野生型菌株Ax2的趋化性。这些结果表明,DIF-1-BODIPY和DIF-1至少部分地通过干扰线粒体活性来诱导茎细胞分化并调节趋化性。