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Taming membranes: functional immobilization of biological membranes in hydrogels.

Kusters I, Mukherjee N, de Jong MR, Tans S, Koçer A, Driessen AJ - PLoS ONE (2011)

Bottom Line: Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active.Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs.This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.

ABSTRACT
Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

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Liposomes containing fluorescent lipid analog DiD (maximum emission at 670 nm) filled with Alexa Fluor 488 (maximum emission at 517 nm) labeled glutathione, (A,B) hydrogel immobilized, (C,D) liposomes in suspension prior immobilization.Images were taken in a Dual-color laser-scanning confocal microscope by scanning an area of 30×30 µM in gel or solution.
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pone-0020435-g002: Liposomes containing fluorescent lipid analog DiD (maximum emission at 670 nm) filled with Alexa Fluor 488 (maximum emission at 517 nm) labeled glutathione, (A,B) hydrogel immobilized, (C,D) liposomes in suspension prior immobilization.Images were taken in a Dual-color laser-scanning confocal microscope by scanning an area of 30×30 µM in gel or solution.

Mentions: Hydrogelator molecules based on 1,3,5-cyclohexyltricarboxamide self-assemble into nano-scaled fibers that form a three dimensional interpenetrating network with defined mesh size [14]. Here, we obtained hydrogels (Figure 1A) by cooling a hot solution (130°C) of 1.3% gelator 1 in buffer to room temperature. For visualization, liposomes of E. coli lipids supplemented with the fluorescent lipid analog DiD were filled with the water-soluble fluorophore Alexa Fluor 488 (AF488) attached to the tripeptide glutathione. Liposomes and proteoliposomes as prepared in this study typically have an average size of around 100 nm as observed by dynamic light scattering and NanoSight particle tracking (data not shown). Immobilization of the filled liposomes was achieved by short vortexing (vigorous shaking) of the liposome suspension in a 1∶1 ratio with the preformed gel. Subsequently, the gel was applied onto a microscopic cover slip followed by a resting phase in which the gel “heals”, i.e. crosslinks between fibers are re-formed leading to a network with incorporated liposomes. The immobilized liposomes were imaged in the gel using a dual color laser scanning confocal microscope. In contrast to the PLs in suspension (Figure 2C and D) which moved while taking the image, the location of the PLs in the hydrogel was entirely stable within the confocal plane (Figure 2A and B). Moreover, the fluorescent signals of the two fluorophores DiD and AF488 co-localize and the level of liposome encapsulated AF488 was similar to that of liposomes prior immobilization (Figure S1). The latter was determined by dual-color fluorescent-burst analysis (DCFBA, for review see [15]), a technique that enables the quantification of co-localizing fluorescent signals and the calculation of relative fluorescence ratios. Using DCFBA, the fluorescent intensities of encapsulated AF488 and DiD of individual liposomes were determined and the ratio of AF488/DiD was calculated for both liposomes diffusing in solution and immobilized in the hydrogel (Figure S1). The average ratio AF488/DiD is an arbitrary unit for the amount of encapsulated dye per liposome and was found to be similar for both conditions (Figure S1). Thus, no significant leakage of AF488 occurred during immobilization in the hydrogel.


Taming membranes: functional immobilization of biological membranes in hydrogels.

Kusters I, Mukherjee N, de Jong MR, Tans S, Koçer A, Driessen AJ - PLoS ONE (2011)

Liposomes containing fluorescent lipid analog DiD (maximum emission at 670 nm) filled with Alexa Fluor 488 (maximum emission at 517 nm) labeled glutathione, (A,B) hydrogel immobilized, (C,D) liposomes in suspension prior immobilization.Images were taken in a Dual-color laser-scanning confocal microscope by scanning an area of 30×30 µM in gel or solution.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105061&req=5

pone-0020435-g002: Liposomes containing fluorescent lipid analog DiD (maximum emission at 670 nm) filled with Alexa Fluor 488 (maximum emission at 517 nm) labeled glutathione, (A,B) hydrogel immobilized, (C,D) liposomes in suspension prior immobilization.Images were taken in a Dual-color laser-scanning confocal microscope by scanning an area of 30×30 µM in gel or solution.
Mentions: Hydrogelator molecules based on 1,3,5-cyclohexyltricarboxamide self-assemble into nano-scaled fibers that form a three dimensional interpenetrating network with defined mesh size [14]. Here, we obtained hydrogels (Figure 1A) by cooling a hot solution (130°C) of 1.3% gelator 1 in buffer to room temperature. For visualization, liposomes of E. coli lipids supplemented with the fluorescent lipid analog DiD were filled with the water-soluble fluorophore Alexa Fluor 488 (AF488) attached to the tripeptide glutathione. Liposomes and proteoliposomes as prepared in this study typically have an average size of around 100 nm as observed by dynamic light scattering and NanoSight particle tracking (data not shown). Immobilization of the filled liposomes was achieved by short vortexing (vigorous shaking) of the liposome suspension in a 1∶1 ratio with the preformed gel. Subsequently, the gel was applied onto a microscopic cover slip followed by a resting phase in which the gel “heals”, i.e. crosslinks between fibers are re-formed leading to a network with incorporated liposomes. The immobilized liposomes were imaged in the gel using a dual color laser scanning confocal microscope. In contrast to the PLs in suspension (Figure 2C and D) which moved while taking the image, the location of the PLs in the hydrogel was entirely stable within the confocal plane (Figure 2A and B). Moreover, the fluorescent signals of the two fluorophores DiD and AF488 co-localize and the level of liposome encapsulated AF488 was similar to that of liposomes prior immobilization (Figure S1). The latter was determined by dual-color fluorescent-burst analysis (DCFBA, for review see [15]), a technique that enables the quantification of co-localizing fluorescent signals and the calculation of relative fluorescence ratios. Using DCFBA, the fluorescent intensities of encapsulated AF488 and DiD of individual liposomes were determined and the ratio of AF488/DiD was calculated for both liposomes diffusing in solution and immobilized in the hydrogel (Figure S1). The average ratio AF488/DiD is an arbitrary unit for the amount of encapsulated dye per liposome and was found to be similar for both conditions (Figure S1). Thus, no significant leakage of AF488 occurred during immobilization in the hydrogel.

Bottom Line: Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active.Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs.This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.

ABSTRACT
Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

Show MeSH
Related in: MedlinePlus