Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance

Zhang, Yanli; Zhang, Xinyan; Kong, Wen-Ru; Wang, Shu‐Qi

doi:10.3390/molecules26206201

Cited by 5 publications

(8 citation statements)

References 63 publications

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“…Only few studies on caveolin-1 expression displayed data on the various forms of the protein and their analyses through Western blots [8]. The oligomers are larger than 250 kDa and found at the interface between the two types of SDS-PAGE gels (concentration and separation) [23,51]. Zhang et al recommended to boil the samples to ensure complete denaturation of such structures prior to SDS-PAGE [23].…”

Section: Discussionmentioning

confidence: 99%

“…The oligomers are larger than 250 kDa and found at the interface between the two types of SDS-PAGE gels (concentration and separation) [23,51]. Zhang et al recommended to boil the samples to ensure complete denaturation of such structures prior to SDS-PAGE [23]. Here, even if the samples were heated at 95°C for 10 min in 4% SDS, the oligomeric forms of caveolin-1 were still present.…”

Section: Discussionmentioning

confidence: 99%

“…In all these systems (except cell-free), the expression of the recombinant caveolin induced the formation of intracellular vesicles within the heterologous host. Notably, in these few expression systems, both whole and truncated versions of caveolin-1 have been demonstrated to be su cient alone to promote membrane budding and protrusion, and the eventual formation of intracellular vesicles presenting high homogeneity of size and shape, if local concentration of the protein is enough [23]. Such intracellular vesicles, called "heterologous caveolae" [17], could be used for diverse biotechnological purposes, and especially for co-expression of other membrane proteins (MPs) of interest [20,24].…”

Section: Introductionmentioning

confidence: 99%

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Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Flourieusse

Bourgeois

Schenckbecker

et al. 2022

Preprint

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Background: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins.Results: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β has been shown to produce caveolin-1β, under its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blotting and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation. Conclusion: These caveolin-1β enriched intracellular neo-formed vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Flourieusse

Bourgeois

Schenckbecker

et al. 2022

Preprint

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show abstract

“…Lipids and lipid mixtures frequently serve as simplified models for cellular membranes [ 176 , 177 , 178 , 179 , 180 ]. However, natural cell membranes are much more complex in their composition and architecture [ 181 ].…”

Section: Applications Of Compartments In the Biomedical Fieldmentioning

confidence: 99%

Current Perspectives on Synthetic Compartments for Biomedical Applications

Heuberger

Korpidou

Eggenberger

et al. 2022

IJMS

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Nano- and micrometer-sized compartments composed of synthetic polymers are designed to mimic spatial and temporal divisions found in nature. Self-assembly of polymers into compartments such as polymersomes, giant unilamellar vesicles (GUVs), layer-by-layer (LbL) capsules, capsosomes, or polyion complex vesicles (PICsomes) allows for the separation of defined environments from the exterior. These compartments can be further engineered through the incorporation of (bio)molecules within the lumen or into the membrane, while the membrane can be decorated with functional moieties to produce catalytic compartments with defined structures and functions. Nanometer-sized compartments are used for imaging, theranostic, and therapeutic applications as a more mechanically stable alternative to liposomes, and through the encapsulation of catalytic molecules, i.e., enzymes, catalytic compartments can localize and act in vivo. On the micrometer scale, such biohybrid systems are used to encapsulate model proteins and form multicompartmentalized structures through the combination of multiple compartments, reaching closer to the creation of artificial organelles and cells. Significant progress in therapeutic applications and modeling strategies has been achieved through both the creation of polymers with tailored properties and functionalizations and novel techniques for their assembly.

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“…In all these systems (except cell-free), the expression of the recombinant caveolin induced the formation of intracellular vesicles within the heterologous host. Notably, in these few expression systems, both whole and truncated versions of caveolin-1 have been demonstrated to be sufficient alone to promote membrane budding and protrusion, and the eventual formation of intracellular vesicles presenting high homogeneity of size and shape, provided local concentration of the protein is high enough [ 23 ]. Such intracellular vesicles, called “heterologous caveolae” [ 17 ], could be used for diverse biotechnological purposes, and especially for co-expression of other membrane proteins (MPs) of interest [ 20 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

et al. 2022

View full text Add to dashboard Cite

Background Caveolae are invaginated plasma membrane domains of 50–100 nm in diameter involved in many important physiological functions in eukaryotic cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins. Results Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β were shown to produce caveolin-1β, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation. Conclusions Lactococcus lactis cells can heterologously overexpress caveolin-1β, generating caveolin-1β enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

show abstract

Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance

Cited by 5 publications

References 63 publications

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Current Perspectives on Synthetic Compartments for Biomedical Applications

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

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