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

Abstract

A 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. The co-stimulation was attained using corresponding ligands: IgE binding antigen and macrophage inflammatory protein 1α (MIP1 α), 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 rearrangement 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).

Figure 1

AFM topographs of six DNP nanostructures: (A) Nano1, (B) Nano2, (C) Nano3, (D) Nano4, (E) Nano5, and (F) Nano6. Inset in (D) shows a zoom-in view. Scale bars are 2 μm and 200nm for images and inset, respectively. All AFM images were acquired under contact mode in ambient condition, with imaging force ranging from 15 to 25 nN.

Figure 2

(A) SEM image of two RBL cells connected by a membrane nanotube. (B1) Zoom-in view of frame 1 indicated in (A). (B2) Zoom-in view of frame 2 indicated in (A).

Figure 3

(A) SEM image of five RBL cells connected in series by membrane nanotubes. (B) SEM image of one RBL cell forming multiple membrane nanotubes with neighboring cells.

Figure 4

SEM images revealing RBL membrane nanotubes after a 1 h interaction with designated nanostructures of haptens in culture media: (A) Nano2, (B) Nano3, (C) Nano4, and (D) Nano6.

Figure 5

(A) Membrane nanotube prevalence and (B) length are plotted as functions of DNP coverage.

Figure 6

Schematic diagram to illustrate DNP nanostructure-induced signaling processes among RBL cells. (A) From the perspective of geometry only, the optimal arrangement of IgE-FcεRI complexes for degranulation. (B) AFM topographic image of DNP nanostructures produced using nanografting.^– The periodicity of this DNP grid nanostructure is 39±4 nm, and the edge-to-edge separation is 22 ± 5 nm. (C) From the perspective of geometry only, arrangements of IgE-FcεRI complexes for discouraging degranulation and facilitating nanotube formation. (D) AFM topographic image of Nano2. Scale bars are as follows: (B) 100 nm; (D) 800 nm.

Figure 7

AFM topography images of (A) SAM1, (B) pure DNP SAMs, and (C) Nano2. SEM images of RBL cells after a 1 h interaction with (D) SAM1, (E) pure DNP SAMs, and (F) Nano2. Scale bars are as follows: (A, B) 25 nm; (C) 800 nm; (D–F) 10 μm.