morange-nano recruitment to disilid functionalized giant unilamellar vesicles (guvs) for generating protein patterns

脂质膜上的光控可逆蛋白质图案

The formation of protein patterns on cell membranes within subcellular regions gives rise to numerous biological processes, including migration, division, and localized cell-to-cell communication1,2. These protein patterns are regulated in space and time, and are highly dynamic. Replicating such protein patterns in synthetic cells is essential for mimicking cellular processes that arise from them and for gaining a better understanding of how such regulation works at a molecular level. Analogous to what is observed for membranes in living cells, methods for generating protein patterns on artificial membranes must capture their dynamics and provide precise spatiotemporal control.
Among various stimuli, light stands out for providing the highest spatiotemporal control and several additional advantages3. Through regulation with light, it is straightforward to illuminate a desired area at any desired time with unmatched precision. In addition, light provides high tunability as both the light intensity and the pulse durations can be adjusted. Furthermore, visible light is harmless to biomolecules, including proteins, and it is possible to even address multiple functionalities with different wavelengths. Hence, light-responsive approaches based on visible light emerge as promising avenues for the controlled and biorthogonal regulation of protein patterns in space and time4,5,6. Utilizing photoswitchable protein pairs from optogenetics, acting as light-inducible dimerizers, provides a straightforward method for recruiting&#160;specific proteins to membranes. In particular, protein patterns have successfully been formed on artificial membranes using the blue light-triggered interaction between iLID (improved light-inducible dimer, based on the photoswitchable LOV2 domain from&#160;Avena sativa) and Nano (wild-type SspB)7,8, the blue light-inducible SpyTag system (BLISS)9, the green light-responsive&#160;protein tetramer CarH10 and the red light-inducible interaction between PhyB and PIF611.
It has been demonstrated that the photoswitchable interaction between iLID&#160;and Nano5 can be used to photo-pattern proteins on model membranes using blue light7. The iLID/Nano interaction is reversible in the dark, highly specific, and operates under physiological conditions. Anchoring iLID onto lipid membrane models, such as giant unilamellar vesicles (GUVs) or supported lipid bilayers (SLBs), enables light-regulated recruitment of Nano to these membranes, which is reversible in the dark. Notably, we observed that introducing a disordered domain to the N-terminus of iLID (resulting in a protein named disiLID) as a tether to a model lipid membrane enhances Nano recruitment efficiency and reversion dynamics8.
By employing the disiLID/Nano interaction, we have developed a method for generating high contrast patterns of Nano-fused proteins of interest (POI) on SLBs and the external membranes of GUVs. This method allows for the creation of protein patterns with remarkable spatial and temporal resolution and high reversibility within minutes. The detailed protocol outlines the process for locally recruiting proteins onto artificial membranes. Specifically, this is achieved by immobilizing a biotinylated version of disiLID on SLBs and GUVs through the biotin-streptavidin (SAv) interaction. Subsequently, fluorescently labeled Nano (mOrange-Nano) is recruited to these disiLID functionalized membranes under blue light illumination. Our experimental protocol offers a straightforward and adaptable approach for achieving localized protein recruitment to membranes. Importantly, this methodology is not confined to the reported SLB and GUV interfaces or mOrange-Nano; it can be extended to other disiLID-functionalized materials and proteins fused to Nano.

作者聚焦：用于人工细胞系统中可逆模式的光可切换蛋白质募集

模型膜上的动态光诱导蛋白质模式

In our work, we aim to reproduce cellular processes in artificial cell-like models that require the precise localization of proteins in a given spot at a certain time. Here, as an example, we demonstrate the formation of dynamic protein patterns on model lipid membranes with high precision and high spatial temporal control using photoswitchable proteins and visible light. One of the current experimental challenge consists of reproducing dynamic protein patterns on model lipid membranes.Here, we need to manipulate these protein patterns fast and reversably with high precision in a biocompatible and non-invasive way. We use photoswitchable proteins to recruit and pattern proteins of interest on supported lipid bilayers and giant unilamellar vesicles. We achieve rapid and localized protein recruitment, and the protein patterning is fully reversible in the dark and can be repeated multiple times.In our protocol, we employ visible light to form protein patterns. Light is an attractive triggers to control interactions with high spatiotemporal resolution and tunability. Light is also not invasive and doesn't damage biomolecules.

mOrange-Nano 募集到 disiLID 功能化巨型单层囊泡 （GUV） 以生成蛋白质模式 

首先，取一管新鲜收获的 GUV。加入链霉亲和素溶液，在室温下静置 30 分钟。然后向 GUV 溶液中加入一微摩尔的 b-disiLID。用铝箔覆盖样品并将其置于黑暗中。用 150 微升 BSA 溶液预处理微载玻片 18 孔玻璃底室 10 分钟。然后取出 BSA 溶液并用 150 微升水清洗孔 3 次。接下来，在缓冲液中加入 145 微升 200 纳摩尔 mOrange-nano 溶液。向溶液中加入 5 微升用 b-disiLID 修饰的 GUV。用铝箔覆盖样品，等待大约 15 分钟让囊泡沉降。然后，将微载玻片置于共聚焦显微镜下。在 552 纳米处激发样品以进行 mOrange 可视化。在 GUV 膜中的 DID 为 638 纳米。mOrange-nano GUVs在黑暗中未能表现出荧光。在 GUV 膜上量化的 mOrange 强度显示蛋白质募集快速有效，且具有完全可逆性。

morange-nano-recruitment-to-disilid-functionalized-giant-unilamellar

Research

JoVE Journal

Bioengineering

mOrange-Nano Recruitment to disiLID Functionalized Giant Unilamellar Vesicles (GUVs) for Generating Protein Patterns

181 次观看.  University of Münster. 首先，取一管新鲜收获的 GUV。加入链霉亲和素溶液，在室温下静置 30 分钟。然后向 GUV 溶液中加入一微摩尔的 b-disiLID。用铝箔覆盖样品并将其置于黑暗中。用 150 微升 BSA 溶液预处理微载玻片 18 孔玻璃底室 10 分钟。然后取出 BSA 溶液并用 150 微升水清洗孔 3 次。接下来，在缓冲液中加入 145 微升 200 纳摩尔 mOrange-nano 溶液。向溶液中加入 5 微升用 b-disiLID 修饰的 GUV。...

视频: mOrange-Nano 募集到 disiLID 功能化巨型单层囊泡 （GUV） 以生成蛋白质模式 

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

mOrange-Nano 募集到 disiLID 功能化巨型单层囊泡 （GUV） 以生成蛋白质模式

mOrange-Nano 募集到 disiLID 功能化巨型单层囊泡（GUV）以生成蛋白质模式