Phosphoinositide Biosensors Kit (Kit #1000000261)

Depositing Lab : Hannes Maib, David Murray

세포 기능에서 막(membrane)의 정체성은 핵심적인 요소이며, 이는 부분적으로  phosphoinositides라는 소수의 인지질 그룹에 의해 결정됩니다. 이들은 inositol headgroups에 인산기가 서로 다르게 결합된 형태를 갖습니다.

addgene은 고정된 세포와 조직에서 phosphoinositide 전체 군을 탐지할 수 있는 재조합 바이오센서 세트를 개발했습니다. 이 방법은 이러한 probes을 세포 내에서 과발현시킬 필요를 없애며, multiplex과 초고해상도 현미경 기법과도 호환됩니다.

이 키트는 실온에서 개별 bacterial stabs 형태로 발송됩니다.

Description

The most common way to visualize the subcellular localization of phosphoinositides is to ectopically overexpress the lipid effectors fused to fluorescent proteins as biosensors (Wills et al., 2018). This approach allows for the detection of membrane identity changes in live cells and has been of immense value over the last decades. However, it also suffers from a major drawback: as these biosensors are generated to have a high affinity, they can all potentially out-compete endogenous effector proteins and thereby perturb the very pathway that is under investigation. In contrast, biosensors with lower affinity and expression levels are themselves out-competed by endogenous effectors. Furthermore, this overexpression-based approach is limited by the ability to deliver DNA into cells or requires time and labor-extensive genomic engineering. It is therefore still a challenge for experimentalists to visualize these critically important lipids, especially in more complex systems such as 3D cell cultures or whole tissues.

One way to circumvent these pitfalls has been to stain for phosphoinositides after fixation and permeabilization using immunocytochemical techniques based on recombinant biosensors (Gillooly et al., 2000; Watt et al., 2002) and antibodies (Hammond et al., 2009; Maekawa and Fairn, 2014; Marat et al., 2017). While this approach is unable to provide dynamic information, it does avoid perturbation of phosphoinositide signaling and does not require overexpression. However, the need for fixatives and detergents requires careful optimizations to avoid artifacts, and the available antibodies require different staining protocols for the visualization of distinct pools of their respective targets (Hammond et al., 2009). As such, there is currently no reliable tool to comprehensively visualize all subcellular phosphoinositide pools with a unifying staining approach.

To address these shortcomings, we have developed recombinant biosensors against all eight phosphoinositides in combination with self-labeling protein tags (SNAP). With the exception of the only known biosensor against PI(5)P, all of these probes are easy to purify using standard bacterial expression systems and demonstrate excellent specificities, as determined using an in vitro supported lipid bilayer approach. Using a single, unified staining protocol, we show that these probes reliably visualize at least six out of the eight phosphoinositide species after fixation and permeabilization. We successfully reproduce the known subcellular localizations of these phosphoinositides and verify the specificity of the staining approach using a range of lipid kinase inhibitors. With the exception of PI(5)P, and with some caveats in detecting PI, this toolkit enables the reliable and reproducible detection of PI(3)P, PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3.

Figure 1: Multiplex staining of phosphoinositides across scales. (a) HeLa cells were fixed, permeabilized, and stained using recombinant biosensors against PI(3)P, PI(4)P, and PI(4,5)P2, conjugated respectively to Alexa488, 546, and 647. (b) NMuMG spheroids were grown in Matrigel and stained with the same combination. (c) Drosophila pupal wings were dissected and stained with the PI(4,5)P2 and PI(4)P biosensors conjugated to Alexa647 and 546, together with Phalloidin conjugated to Alexa488, to visualize the actin cytoskeleton and cellular junctions.

Original Publication

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides. Maib H, Adarska P, Hunton R, Vines JH, Strutt D, Bottanelli F, Murray DH. J Cell Biol. 2024 Jun 3;223(6):e202310095. doi: 10.1083/jcb.202310095. Epub 2024 Apr 5. PubMed  Article 

Contents of Kit