Lipid transport between organelles is essential for many biological processes and usually proceeds at membrane contact sites via lipid transfer proteins (LTPs). This process is challenging to study as most current approaches for tracking intracellular lipid transport are indirect, and it is unclear if the insights obtained in vitro are translatable to cellular settings. To address this, Luan, Liang et al. developed a method for imaging lipid transport between the endoplasmic reticulum and the plasma membrane. The method was based on repurposing the IMPACT (imaging PLD activity with clickable alcohols via transphosphatidylation) technique, initially developed for visualizing phospholipase D (PLD) activity. An oxo-transcyclooctene primary alcohol probe is used by PLD for transphosphatidylation, resulting in the formation of oxo-transcyclooctene-containing phosphatidyl alcohol lipids at the plasma membrane. These lipids are tagged by a bioorthogonal reaction with a fluorogenic tetrazine−BODIPY reagent to obtain fluorescent phosphatidyl alcohol lipids, which translocate to the endoplasmic reticulum and can be visualized by fluorescence imaging. The team then used short interfering RNA (siRNA) to obtain triple knockdown of extended synaptotagmins (E-Syts) and showed that these LTPs are involved in the interorganelle transport of IMPACT lipids. Additionally, the transport could be redirected to mitochondria using E-Syt-based artificial tethers at endoplasmic reticulum–mitochondria contact sites. The team was also able to reconstitute the transport of IMPACT lipids between two liposomes in vitro, confirming that it requires E-Syts. The IMPACT-based method could potentially be used to monitor the activity of other LTPs and obtain new insights into lipid transport within cells.

Original reference: ACS Chem. Biol. https://doi.org/10.1021/acschembio.4c00345 (2024)

细胞器之间的脂质运输对于许多生物过程至关重要，通常通过脂质转移蛋白 (LTP) 在膜接触位点进行。这一过程的研究具有挑战性，因为目前大多数追踪细胞内脂质转运的方法都是间接的，并且尚不清楚体外获得的见解是否可以转化为细胞环境。为了解决这个问题，Luan，Liang 等人。开发了一种对内质网和质膜之间的脂质运输进行成像的方法。该方法基于对 IMPACT（通过转磷脂酰化使用可点击醇成像 PLD 活性）技术的重新利用，该技术最初是为可视化磷脂酶 D (PLD) 活性而开发的。 PLD 使用氧代-反式环辛烯伯醇探针进行转磷脂酰化，导致在质膜上形成含有氧代-反式环辛烯的磷脂酰醇脂质。这些脂质通过与荧光四嗪-BODIPY试剂的生物正交反应进行标记，以获得荧光磷脂酰醇脂质，其易位至内质网并且可以通过荧光成像可视化。然后，研究小组使用短干扰 RNA (siRNA) 获得了扩展突触结合蛋白 (E-Syts) 的三重敲低，并表明这些 LTP 参与了 IMPACT 脂质的细胞器间转运。此外，可以在内质网-线粒体接触位点使用基于 E-Syt 的人工系绳将运输重定向至线粒体。该团队还能够在体外重建两个脂质体之间的 IMPACT 脂质运输，证实它需要 E-Syts。基于 IMPACT 的方法有可能用于监测其他 LTP 的活性，并获得对细胞内脂质运输的新见解。

Original reference: ACS Chem. Biol. https://doi.org/10.1021/acschembio.4c00345 (2024)