ACS Nano. 2015 Jul 28;9(7):6738-46.

Engineered Nanostructures of Haptens Lead to Unexpected Formation of Membrane Nanotubes Connecting Rat Basophilic Leukemia Cells

 Jie-Ren Li,1 Shailise S. Ross,1 Yang Liu,1 Ying X. Liu,1 Kang-Hsin Wang,1 Huan-Yuan Chen,2,3 Fu-Tong Liu,2,3 Ted A. Laurence4 and Gang-yu Liu1*

1 Department of Chemistry, University of California, Davis, California, 95616

2Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, California, 95817

3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC

4Lawrence Livermore National Laboratory, Livermore, CA 94550




Recent finding reports that co-stimulation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) and the chemokine receptor 1 (CCR1) triggered formation of membrane nanotubes among bone marrow-derived mast cells (BMMCs). The co-stimulation was attained using corresponding ligands: IgE binding antigen and MIP-1α, respectively. However, this approach failed to trigger formation of nanotubes among Rat basophilic leukemia (RBL) cells due to the lack of CCR1 on the cell surface (Int. Immunol. 2010, 22, (2), 113-128). RBL cells are frequently used as a model for mast cells, and are best known for antibody-mediated activation via FcεRI. This work reports the successful formation of membrane nanotubes among RBLs using only one stimulus, a hapten of 2,4-dinitrophenyl (DNP) molecules, which are presented as nanostructures with our designed spatial arrangements. This observation underlines the significance of the local presentation of ligands in the context of impacting the cellular signaling cascades. In the case of RBL, certain DNP nanostructures suppress antigen-induced degranulation and facilitate the re-arrangement of the cytoskeleton to form nanotubes. These results demonstrate an important scientific concept; engineered nanostructures enable cellular signaling cascades, where current technologies encounter great difficulties. More importantly, nanotechnology offers a new platform to selectively activate and/or inhibit desired cellular signaling cascades.

KEYWORDS: atomic force microscopy (AFM); haptens; mast cells; membrane nanotubes; particle lithography; rat basophilic leukemia (RBL) cells; scanning electron microscopy (SEM)

PMID: 26057701



Nature taught us local environment greatly impacts the behavior and function of living cells, a well-known example of which is the importance of extracellular matrices (ECM).1-5 The mechanisms frequently involve the interactions among ligands and cellular membrane receptors, which guide the clustering of receptors, as such impact on the downstream protein complex formation, and cellular signaling processes.6,7 Therefore, by arranging ligands in designated arrangements one could, in principle, regulate or control the cellular signaling processes, behavior and function. Arranging ligands by design requires state-of-the-art nanotechnology, as the size and arrangement of ligand-receptor interaction and clustering are at nanometer scale.8-16 Using a model antigen as ligand, 2, 4-dinitrophenyl (DNP), this work demonstrates the power and impact of arranging ligand at nanometer scale on cellular behavior of mast cells, such as rat basophilic leukemia cells (RBL).


Figure 1 compares a conventional method to produce surface presented ligands with the nanoarrays of ligands arranged by design using nanotechnology. Conventional means of producing surface bound ligands typically follow various “mix-and-deposit” protocols. One example of using a mixed self-assembled monolayers of DNP terminal thiol and octadecanethiol is shown in Figure 1A, where the DNP termini randomly arrange on surfaces with separation ranging from 36 nm to 0.80 mm.16,17 Exposing this ECM mimetic surface to culture medium solution containing RBL cells results in adhesion of RBL on surfaces. As shown in Figure 1D and 1G, some membrane show ridges but most exhibit a relatively smooth morphology, indicating that cells were in a resting or non-activated state.18,19 Using nanografting, an atomic force microscopy (AFM) based nanolithography method, nanogrids of DNP were produced on surfaces. One of the nanogrids is shown in Figure 1B with periodicity of 39 ± 4 nm and line width of 17.1 ± 1.7 nm.16 Upon exposure to RBL cells, fast and high degrees of activation were observed, as evidenced by cellular spreading, and membrane ridges (Figure 1E and 1H). Changing the spatial arrangement at nanometer scale could alter the outcome dramatically. By forming arrays of nanodonuts made of DNP molecules, the nanostructures-cell interactions were measured and compared to those of the nanogrids. One of the nanodonuts arrays was formed by particle lithography.20-23 and its structure is shown in Figure 1C, where the periodicity is 807 ± 10 nm, while ring width and inner diameter are 127 ± 7 nm, and 153 ± 6 nm, respectively. These engineered nanostructures were then immersed in Dulbecco’s modified Eagle medium (DMEM) cell media containing RBL cells pre-sensitized by anti-DNP antibody, namely immunoglobulin E (IgE). After 1 h soaking, membrane nanotubes were observed among nearest neighbor RBL cells under scanning electron microscopy (SEM) imaging.17 The single membrane nanotube shown in Figure 1I measures 6.75 μm long and 76 nm in diameter, which falls in range with known membrane nanotubes from other cells types such as pheochromocytoma cell line (PC12).24 Membrane connected multiple cells were also formed as shown in Figure 1F, where one RBL cell in the center formed multiple intercellular membrane nanotubes connected with three surrounding cells.



Figure 1. AFM topography images of [A] a binary self-assembled monolayer (SAM) of DNP thiol:octadecanelthiol = 1:20; [B] nanogrids of DNP; [C] nanorings of DNP. Scale bars in [A]–[C] = 400 nm. [D], [E] and [F] are SEM images of RBL cells after 1 h incubation on the SAM, nanogrids and nanodonuts, respectively. Scale bars in [D]-[F] = 20 μm. [G] and [H] represent the zoom-in view as defined in [D] and [E], respectively. [I] SEM image from the same sample as [F] revealing two RBL cells connected by a membrane nanotube. Scale bars in [G]–[ I] = 10 μm.


The formation of membrane nanotubes among RBL cells was unexpected, because formation of membrane nanotubes requires the co-stimulation of the two receptors FcεRI and CCR1.25 However, RBL cells are CCR1 deficient.25 The most well-known signaling cascades for RBL cells are antigen mediated degranulation, or activation, which is important to allergy.6,7,18 As shown in Figure 2, the initial steps involve FcεRI receptors bound to antigens to form clusters, which causes degranulation to release histamine downstream. The membrane exhibit rough morphology, with characteristic ridges, from SEM investigations.9,16,18,19 The nanogrids shown in Figure 1B were designed to foster the closely packed FcεRI clusters (20 nm separation among nearest neighbors).16 All nanorings produced are well separated by >305 nm, which inhibit the clustering of FcεRI receptors, and force the less-known pathways of M-Sec mediated membrane nanotube formation.6,26-29




Figure 2. Schematic diagram to illustrate how various designs of DNP nanostructures may impact the signaling processes among RBL cells.



Membrane nanotubes provide membrane continuity among connected cells and enable intercellular exchange of both membrane carrying molecules and cytoplasmatic content. They play vital roles in many physiological processes including immune defense, tumorigenesis, transmission of pathogens, and cell differentiation. Membrane nanotubes have been observed among various cell types, such as pheochromocytoma cell line, nature killer cells, dendritic cells, and T cells. Our finding of membrane nanotube formation among immune cells sparks much interest in regulation of immunological processes. More importantly, our work demonstrates that nanotechnology offers a new platform to selectively activate and inhibit desired cellular signaling cascades.



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