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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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Opening of the MscL channel monitored on a single hydrogel immobilized GUV supplemented with the fluorescent lipid analog DiD.(A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).
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pone-0020435-g003: Opening of the MscL channel monitored on a single hydrogel immobilized GUV supplemented with the fluorescent lipid analog DiD.(A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).

Mentions: To investigate the suitability of the hydrogel for immobilization of very large and fragile membrane systems, we prepared several micron sized GUVs and tested them for membrane integrity and membrane protein activity in the gel. The performance of a hydrogel as described in the previous section was tested for GUV immobilization and monitoring of membrane protein activity. The mechanosensitive channel of large conductance, MscL, from E. coli was reconstituted into liposomes and the resulting PLs were used to generate DiD labeled GUVs [16]. MscL is a bacterial membrane channel protein that senses the increase in the lateral pressure in the membrane due to a hypo-osmotic shock. It functions as a safety valve to release the turgor pressure by opening a large, non-selective pore and releasing molecules and even peptides up to 6.5 kDa [17], [18]. Here we used an engineered version of this channel, MscLG22C [19], [20]. MscLG22C can be activated by labeling the cysteine at position22 with the charged molecule [2-(trimethylammonium)ethyl]methane thiosulfonate bromide (MTSET). This covalent modification results in the opening and closing of the channel in the absence of its native trigger, i.e. membrane tension [21]. The performance of MscL, embedded in hydrogel immobilized GUVs, was tested by following the triggered influx of a fluorescent dye (calcein) into GUVs through MscL using confocal microscopy. For incorporation of GUVs into the gel, we took advantage of the inherent reversibility of the physical crosslinks that hold the gel together. By vigorous shaking on a vortex mixer, crosslinks between and within fibers are broken, resulting in a viscous suspension. Due to their fragile nature, DiD labeled GUVs containing MscL were embedded in a hydrogel of gelator 1 by first liquefying the preformed hydrogel by vigorous shaking it for 1 min and mixing the hydrogel afterwards with 0.5 volume of GUVs using a pipette. The gel-GUV mixture was applied on a cover slip and incubated for about 10 minutes to ensure gel healing. During this time period, new crosslinks between the fibers are formed and short fibers can assemble and form longer fibers. The DiD stained membrane of an approximately 10 µm sized MscL-GUV was imaged by a two-dimensional scan using a dual laser confocal microscope (Figure 3A, lower panel). Calcein, added to the hydrogel prior to imaging without disturbing the setup, is surrounding the GUVs (Figure 3A, upper panel, first picture). Since MscL was not activated at this point, it stayed closed and calcein could not penetrate into the GUV demonstrating the membrane integrity. Upon the addition of the membrane-impermeable channel activator, MTSET, the channel opened and the gradual time dependent filling up of the GUV was observed (Figure 3A–C). The concentration of calcein inside and outside of the GUV was quantified by a one dimensional cross-section through the center of the GUV at the different time points illustrating MscL channel opening (Figure 3B). Time dependent influx of calcein into the GUV was quantified measuring the calcein fluorescence at the center of the GUV resulting in a kinetic saturating curve (Figure 3C). Equilibration of the calcein concentrations inside and outside of the GUV was reached at around 180 s (Figure 3C), in accordance with the results obtained with PLs in solution (data not shown). Upon addition of buffer containing MTSET, a slight shift in the focal plane was observed resulting from a minor movement of the gel such that a smaller cross-section of the GUV was imaged. As a control, buffer without MTSET was added to MscL GUVs in another gel (Figure S2). No influx of calcein into the GUV could be observed (Figure 3C and S2B) demonstrating the stability of the GUV and the closed state of the MscL channel. Taken together, this data demonstrates that MscL, a mechanosensitive membrane protein, in GUVs is not affected by the hydrogel, which allows single molecule measurements. Moreover, different ways of vesicle immobilization are possible and ensure membrane integrity even of fragile, micron sized vesicles.


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)

Opening of the MscL channel monitored on a single hydrogel immobilized GUV supplemented with the fluorescent lipid analog DiD.(A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).
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pone-0020435-g003: Opening of the MscL channel monitored on a single hydrogel immobilized GUV supplemented with the fluorescent lipid analog DiD.(A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).
Mentions: To investigate the suitability of the hydrogel for immobilization of very large and fragile membrane systems, we prepared several micron sized GUVs and tested them for membrane integrity and membrane protein activity in the gel. The performance of a hydrogel as described in the previous section was tested for GUV immobilization and monitoring of membrane protein activity. The mechanosensitive channel of large conductance, MscL, from E. coli was reconstituted into liposomes and the resulting PLs were used to generate DiD labeled GUVs [16]. MscL is a bacterial membrane channel protein that senses the increase in the lateral pressure in the membrane due to a hypo-osmotic shock. It functions as a safety valve to release the turgor pressure by opening a large, non-selective pore and releasing molecules and even peptides up to 6.5 kDa [17], [18]. Here we used an engineered version of this channel, MscLG22C [19], [20]. MscLG22C can be activated by labeling the cysteine at position22 with the charged molecule [2-(trimethylammonium)ethyl]methane thiosulfonate bromide (MTSET). This covalent modification results in the opening and closing of the channel in the absence of its native trigger, i.e. membrane tension [21]. The performance of MscL, embedded in hydrogel immobilized GUVs, was tested by following the triggered influx of a fluorescent dye (calcein) into GUVs through MscL using confocal microscopy. For incorporation of GUVs into the gel, we took advantage of the inherent reversibility of the physical crosslinks that hold the gel together. By vigorous shaking on a vortex mixer, crosslinks between and within fibers are broken, resulting in a viscous suspension. Due to their fragile nature, DiD labeled GUVs containing MscL were embedded in a hydrogel of gelator 1 by first liquefying the preformed hydrogel by vigorous shaking it for 1 min and mixing the hydrogel afterwards with 0.5 volume of GUVs using a pipette. The gel-GUV mixture was applied on a cover slip and incubated for about 10 minutes to ensure gel healing. During this time period, new crosslinks between the fibers are formed and short fibers can assemble and form longer fibers. The DiD stained membrane of an approximately 10 µm sized MscL-GUV was imaged by a two-dimensional scan using a dual laser confocal microscope (Figure 3A, lower panel). Calcein, added to the hydrogel prior to imaging without disturbing the setup, is surrounding the GUVs (Figure 3A, upper panel, first picture). Since MscL was not activated at this point, it stayed closed and calcein could not penetrate into the GUV demonstrating the membrane integrity. Upon the addition of the membrane-impermeable channel activator, MTSET, the channel opened and the gradual time dependent filling up of the GUV was observed (Figure 3A–C). The concentration of calcein inside and outside of the GUV was quantified by a one dimensional cross-section through the center of the GUV at the different time points illustrating MscL channel opening (Figure 3B). Time dependent influx of calcein into the GUV was quantified measuring the calcein fluorescence at the center of the GUV resulting in a kinetic saturating curve (Figure 3C). Equilibration of the calcein concentrations inside and outside of the GUV was reached at around 180 s (Figure 3C), in accordance with the results obtained with PLs in solution (data not shown). Upon addition of buffer containing MTSET, a slight shift in the focal plane was observed resulting from a minor movement of the gel such that a smaller cross-section of the GUV was imaged. As a control, buffer without MTSET was added to MscL GUVs in another gel (Figure S2). No influx of calcein into the GUV could be observed (Figure 3C and S2B) demonstrating the stability of the GUV and the closed state of the MscL channel. Taken together, this data demonstrates that MscL, a mechanosensitive membrane protein, in GUVs is not affected by the hydrogel, which allows single molecule measurements. Moreover, different ways of vesicle immobilization are possible and ensure membrane integrity even of fragile, micron sized vesicles.

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