preparation of giant unilamellar vesicles (guvs) for fabricating light-regulated reversible protein patterns

Lipid Membranlar Üzerinde Işık Kontrollü Tersinir Protein Deseni

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.

Yazar Spotlight: Yapay Hücresel Sistemlerde Tersinir Modelleme için Fotoğraf Değiştirilebilir Protein Alımı

Model Membranlarda Dinamik Işık Kaynaklı Protein Modelleri

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.

Işık Ayarlı Tersinir Protein Kalıpları Üretmek için Dev Unilamellar Veziküllerin (GUV'ler) Hazırlanması

Başlamak için, Ultra saf suda 100 milimolar sükroz içeren% 5'lik bir polivinil alkol çözeltisi hazırlayın. 400 RPM'de sallarken gece boyunca 80 santigrat derecede karıştırın. Daha sonra istenen bileşime sahip kloroformda bir lipit çözeltisi hazırlayın.Hazırlamak için GUV'ler, hazırlanan polivinil alkol çözeltisinden 40 mikrolitrelik ince bir tabakayı bir cam slayt üzerine yaymak için bir pipet ucu kullanır. Bu tabakayı 50 santigrat derecede 30 dakika kurutun. Daha sonra bir iğne kullanarak polivinil alkol tabakasına beş mikrolitre lipit çözeltisi yayın ve bir saat boyunca 30 santigrat derecede kurumaya bırakın.Şimdi, işlevselleştirilmiş cam slayt üzerine bir oda monte etmek için bir ara parça ve ikinci bir cam slayt kullanın, odaya bir mililitre rehidrasyon tamponu ekleyin ve GUV'ler oluşturmak için oda sıcaklığında bir saat inkübe edin. Ardından hazneyi ters çevirin ve cam yüzeylere hafifçe vurun. İnşa edilmiş hazneyi açmak için bir taraftaki cam sürgüyü dikkatlice çıkarın ve GUV'leri bir pipetle hasat edin.Solüsyonu plastik bir tüpe yerleştirin ve GUV'lerin iki saat oturmasına izin verin.

preparation-giant-unilamellar-vesicles-guvs-for-fabricating-light

Research

JoVE Journal

Bioengineering

Preparation of Giant Unilamellar Vesicles (GUVs) for Fabricating Light-Regulated Reversible Protein Patterns

268 Görüntüleme.  University of Münster. Başlamak için, Ultra saf suda 100 milimolar sükroz içeren% 5'lik bir polivinil alkol çözeltisi hazırlayın. 400 RPM'de sallarken gece boyunca 80 santigrat derecede karıştırın. Daha sonra istenen bileşime sahip kloroformda bir lipit çözeltisi hazırlayın.Hazırlamak için GUV'ler, hazırlanan polivinil alkol çözeltisinden 40 mikrolitrelik ince bir tabakayı bir cam slayt üzerine yaymak için bir pipet ucu kullanır. Bu tabakayı 50 santigrat derecede 30 dakika kurutun....

Video: Işık Ayarlı Tersinir Protein Kalıpları Üretmek için Dev Unilamellar Veziküllerin (GUV'ler) Hazırlanması

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).