Department of Biochemistry and Molecular Biology, East Carolina Diabetes and Obesity Institute, East Carolina University, NC 27834, USA.
J Nutr Biochem. 2013 Jan;24(1):188-95. doi: 10.1016/j.jnutbio.2012.04.010. Epub 2012 Jul 26.
Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo.
二十二碳六烯酸 (DHA) 在体外和体内破坏质膜脂质微区的大小和有序性。然而，DHA 的高度无序结构如何通过机制适应来增加紧密堆积的脂质微区的有序性尚不清楚。因此，我们研究了一种新型的 DHA-Bodipy 荧光探针来解决这个问题。我们首先确定 DHA-Bodipy 探针是否定位于原代 B 细胞和永生化 EL4 细胞的质膜上。图像分析显示，DHA-Bodipy 比 EL4 细胞更有效地定位于原代 B 细胞的质膜上。然后，我们确定该探针是否检测到质膜有序性的变化。对延时电影的定量分析表明，DHA-Bodipy 对膜分子有序性敏感。这使我们能够研究 DHA-Bodipy 如何适应有序的脂质微区。为此，我们在具有不同组成的脂质体中进行了稳态和时间分辨荧光各向异性测量。与细胞培养研究相似，探针对脂质体中的膜有序性高度敏感。此外，这些实验表明，与对照相比，当探针掺入高度有序的微区时，其荧光寿命和分子有序性增加。此外，与对照相比，探针的旋转扩散显著降低。总之，DHA-Bodipy 对膜有序性非常敏感，并首次表明，尽管 DHA 具有柔韧性，但它可以在有序的脂质微区内部减少旋转运动而变得有序。从机制上讲，这解释了 DHA 酰基链如何在体内形成脂质微区时增加有序性。
