Peptide-targeted delivery of a pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei.
Peptide-targeted delivery of a pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei.
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DOI：
10.1021/bi400029m
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发表时间：
2013-05-28
期刊：
影响因子：
2.9
通讯作者：
Christensen KA
中科院分区：
文献类型：
--
作者：
Lin S;Morris MT;Ackroyd PC;Morris JC;Christensen KA
对原生动物寄生虫布氏锥虫细胞器动态变化的研究一直有限，部分原因是将分析探针靶向特定亚细胞区室存在困难。在此，我们展示了一种用于布氏锥虫糖质体中pH定量的比率型探针的应用。该探针由编码过氧化物酶体靶向序列（F - PTS1，乙酰 - CKGGAKL）的肽段与对pH有响应的荧光素偶联而成。当与活的寄生虫一起孵育时，探针在囊泡结构内被内化，这些囊泡结构与糖质体标记物共定位。在4°C下抑制F - PTS1的摄取以及与荧光葡聚糖的脉冲追踪共定位表明，探针最初是通过非受体介导的内吞作用被摄取的，但随后与葡聚糖分开运输，并在糖质体周转过程中标记的糖质体和溶酶体最终融合之前定位于糖质体内部。在布氏锥虫糖质体中使用F - PTS1进行的细胞器内测量和pH校准表明，生理条件下静止的糖质体pH为7.4 ± 0.2。然而，在缺乏葡萄糖的缓冲液中孵育会在20分钟内引发糖质体的轻度酸化，最终观察到的pH为6.8 ± 0.3。重新引入葡萄糖可逆转这种糖质体酸化。将比率型荧光传感器和报告分子与PTS肽偶联为监测糖质体对环境条件变化的原位反应提供了一种极有价值的工具，并且可应用于其他动基体目寄生虫。
Studies of dynamic changes in protozoan parasite Trypanosoma brucei organelles have been limited, in part because of the difficulty of targeting analytical probes to specific subcellular compartments. Here we demonstrate application of a ratiometric probe for pH quantification in T. brucei glycosomes. The probe consists of a peptide encoding the peroxisomal targeting sequence (F-PTS1, acetyl-CKGGAKL) coupled to fluorescein, which responds to pH. When incubated with living parasites, the probe is internalized within vesicular structures that co-localize with a glycosomal marker. Inhibition of uptake of F-PTS1 at 4°C and pulse-chase co-localization with fluorescent dextran suggested that the probe is initially taken up by non-receptor-mediated endocytosis, but is subsequently trafficked separately from dextran and localized within glycosomes, prior to final fusion of labeled glycosomes and lysosomes as part of glycosomal turnover. Intra-organellar measurements and pH calibration with F-PTS1 in T. brucei glycosomes indicate that the resting glycosomal pH under physiological conditions is 7.4 ± 0.2. However, incubation in glucose-depleted buffer triggered mild acidification of the glycosome over a period of 20 min, with a final observed pH of 6.8 ± 0.3. This glycosomal acidification was reversed by reintroduction of glucose. Coupling of ratiometric fluorescent sensors and reporters to PTS peptides offers an invaluable tool for monitoring in situ glycosomal response(s) to changing environmental conditions and could be applied to additional kinetoplastid parasites.
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影响因子：
1.5
作者：
COPPENS, I;BAUDHUIN, P;COURTOY, PJ
通讯作者：
COURTOY, PJ
影响因子：
4.8
作者：
Jankowski, A;Kim, JH;Grinstein, S
通讯作者：
Grinstein, S
DOI：
10.1111/j.1550-7408.1975.tb00943.x
发表时间：
1975-01-01
期刊：
JOURNAL OF PROTOZOOLOGY
影响因子：
--
作者：
LANGRETH, SG;BALBER, AE
通讯作者：
BALBER, AE
影响因子：
4.8
作者：
Dodson, Heidi C.;Morris, Meredith T.;Morris, James C.
通讯作者：
Morris, James C.
影响因子：
4
作者：
Costa-Pinto, D;Trindade, LS;Traub-Cseko, YM
通讯作者：
Traub-Cseko, YM