Modulation of In-Membrane Receptor Clustering upon Binding of Multivalent Ligands

Grochmal, Anna; Ferrero, Elena; Milanesi, Lilia; Tomàs, Salvador

doi:10.1021/ja404428u

Cited by 33 publications

(35 citation statements)

References 37 publications

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“…The data suggest that preclustering of the receptor ligands (in this case, biotin anti-CD95) on oil drops is directly correlated to the effectiveness of inducing apoptosis through the CD95 receptor. This result is consistent with the known effect of the CD95 receptor clustering on apoptosis (30)(31)(32)(33).…”

Section: Resultssupporting

confidence: 91%

“…Conjugation of antibodies and ectodomains on nanoparticles (34) and liposomes through chemical reactions (35) or affinity tags (27) intends to mimic the cell surface, but the orientation and position of chemically conjugated protein ligands can be difficult to control. It is not possible to form extensive clusters on nanoparticles and liposomes that can potentially induce strong biological responses (32,33). In contrast, ssMPs are more likely to orient properly on oil drops, capable of being spatially clustered, and in a fluidic environment that facilitates the communication with receptors on cell surfaces.…”

Section: Resultsmentioning

confidence: 99%

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One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

Yunker

Asahara

Hung

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell-cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water-oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.embrane proteins populate the surfaces of cells and allow cells to sense and interact with their external environments. Membrane proteins constitute 25-30% of all proteins identified in sequenced genomes, and understanding their functions is important, because they represent the majority of current drug targets (1). Conventionally, function is inferred from structural studies of membrane proteins; these studies involve expression and purification of natural or recombinant membrane proteins followed by crystallization. However, membrane proteins are notoriously hard to work with because of their hydrophobic domains that can cause misfolding and aggregation (2). Consequently, detergents and membrane mimetics (3) are used to solubilize membrane proteins. To avoid cytotoxicity caused by in vivo expression and enable highthroughput production, cell-free systems are used to express membrane proteins in vitro (4-7). Despite recent progresses, understanding the functions of membrane proteins is still a lengthy and difficult process.Here, we develop a one-pot approach for synthesis, assembly, and display of single-span membrane proteins (ssMPs) for direct functional studies. ssMPs contain a single transmembrane (TM) helix that anchors the hydrophilic ectodomains on the membrane surface; ssMPs represent approximately one-half of all membrane proteins and are involved ...

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

Yunker

Asahara

Hung

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In living cells, phenomena such as signal transduction, which are fundamental for immunological responses and neurotransmission, are oen associated with the formation of domains and the clustering of receptors at a cell surface. [1][2][3] Multivalent ligand-receptor interactions at the interface, in combination with the uidity of the cell membrane, promote the rearrangement and co-localization of receptors on a surface, and thereby govern these biological responses. 4 The molecular processes of binding, diffusion and clustering occur at characteristic length and time scales, and these can be distinctly different from those associated with the biological processes that they induce.…”

Section: Introductionmentioning

confidence: 99%

Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles

Iorio

Meulman

et al. 2020

Chem. Sci.

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The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a better understanding of biomolecular processes, such as the recruitment of receptors at interfaces, at the molecular level. Here we report a model system based on supported lipid bilayers (SLBs) for the investigation of the clustering of receptors at their interface. Biotinylated SLBs, used as cell membrane mimics, were functionalized with streptavidin (SAv), used here as receptor. Subsequently, biotinylated small (SUVs) and giant (GUVs) unilamellar vesicles were bound to the SAv-functionalized SLBs by multivalent interactions and found to induce the recruitment of both SAv on the SLB surface and the biotin moieties in the vesicles. The recruitment of receptors was investigated with quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed the identification of the biotin and SAv densities necessary to obtain receptor recruitment. At approx. 0.6% of biotin in the vesicles, a transition between dense and low vesicle packing was observed, which coincided with the transitions between recruitment in the vesicles vs. recruitment in the SLB and between full and partial use of the biotin moieties in the vesicle. Direct optical visualization of the clustering at the interface of individual GUVs with the SLB platform was achieved with fluorescence microscopy, showing recruitment of SAv at the contact area as well as the deformation of the vesicles upon binding. Different vesicle binding regimes were observed for lower and higher biotin densities in the vesicles and at the SLBs. A more quantitative analysis of the molecular parameters implied in the interaction, indicated that approx.10% of the vesicle area constitutes the contact area. Moreover, the SUV binding and recruitment appeared to be fast on the analysis time scale, whereas the binding of GUVs is slower due to the larger SLB area over which SAv recruitment needs to occur. The mechanisms revealed in this study may provide insight in biological processes in which recruitment occurs. † Electronic supplementary information (ESI) available: DLS, FRAP and QCM measurements, uorescence microscopy images and calculation details. See

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“…If clustering is taken as the rate-limiting step in cellular uptake kinetics, then it follows, logically, that the greater the residence time on the membrane the higher the probability to form NP clusters and consequently the greater the uptake. 43,44 Indeed, more GNRs were found adjacent to the membrane for the longest nanorod than any of the shorter ones. These observations are in close agreement with the predictions of Gao et al who showed that the increased uptake of larger NPs, or clusters, is due to the high membrane bending energies associated with the endocytosis of smaller NPs.…”

Section: Comparison With Modelsmentioning

confidence: 99%

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells

et al. 2016

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The impact of nanoparticles (NPs) upon biological systems can be fundamentally associated with their physicochemical parameters. A further often-stated tenet is the importance of NP shape on rates of endocytosis. However, given the convoluted parameters concerning the NP-cell interaction, it is experimentally challenging to attribute any findings to shape alone. Herein we demonstrate that shape, below a certain limit, which is specific to nanomedicine, is not important for the endocytosis of spherocylinders by either epithelial or macrophage cells in vitro. Through a systematic approach, we reshaped a single batch of gold nanorods into different aspect ratios resulting in near-spheres and studied their cytotoxicity, ( pro-)inflammatory status, and endocytosis/exocytosis. It was found that on a length scale of ∼10-90 nm and at aspect ratios less than 5, NP shape has little impact upon their entry into either macrophages or epithelial cells. Conversely, nanorods with an aspect ratio above 5 were preferentially endocytosed by epithelial cells, whereas there was a lack of shape dependent uptake following exposure to macrophages in vitro. These findings have implications both in the understanding of nanoparticle reshaping mechanisms, as well as in the future rational design of nanomaterials for biomedical applications.

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Modulation of In-Membrane Receptor Clustering upon Binding of Multivalent Ligands

Cited by 33 publications

References 37 publications

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells

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