Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts.

氧固醇是胆固醇代谢物，在控制细胞稳态方面具有多种功能。特别是，27-羟基胆固醇 (27-OH-Chol) 已被证明可以调节多种生理功能，但对其摄取、细胞内运输和细胞外排知之甚少。这主要是由于缺乏合适的 27-OH-Chol 类似物，它们与这种氧甾醇非常相似。在这里，我们展示了固有荧光的 27-羟基胆甾三烯醇 (27-OH-CTL)，它与 ​​27-OH-Chol 的不同之处仅在于在类固醇环系统中具有两个额外的双键。基于分子动力学 (MD) 模拟，我们表明与 27-OH-Chol 相比，27-OH-CTL 具有几乎相同的膜特性。通过对活细胞中的 27-OH-CTL 和胆固醇类似物胆甾三烯醇 (CTL) 进行比较成像，我们评估了单个羟基对甾醇贩运的影响。我们发现人类成纤维细胞比 27-OH-CTL 吸收更多的 CTL，但更有效地流出氧甾醇类似物。对于这两种甾醇，流出包括囊泡从质膜脱落。细胞内，27-OH-CTL 主要在脂滴 (LD) 中积累，而 CTL 主要存在于内体和溶酶体中。使用光漂白后的荧光恢复 (FRAP)，我们发现两种甾醇都有一个快速交换池，其移动速度比含有小泡和 LD 的甾醇快几个数量级。总之，通过应用 27-OH-Chol 的新荧光衍生物，我们证明人类细胞可以基于侧链中的单个羟基来区分甾醇，从而导致不同的运输路线、动力学和流出动力学。