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. 2005 Nov 8;21(23):10693-8.
doi: 10.1021/la051243d.

Neuronal activation by GPI-linked neuroligin-1 displayed in synthetic lipid bilayer membranes

Michael M Baksh  1 , Camin Dean, Sophie Pautot, Shannon DeMaria, Ehud Isacoff, Jay T Groves
Affiliations

Neuronal activation by GPI-linked neuroligin-1 displayed in synthetic lipid bilayer membranes

Michael M Baksh et al. Langmuir. .

Abstract

We have characterized, in vitro, interactions between hippocampal neuronal cells and silica microbeads coated with synthetic, fluid, lipid bilayer membranes containing the glycosylphosphatidyl inositol (GPI)-linked extracellular domain of the postsynaptic membrane protein neuroligin-1. These bilayer-neuroligin-1 beads activated neuronal cells to form presynaptic nerve terminals at the point of contact in a manner similar to that observed for live PC12 cells, ectopically expressing the full length neuroligin-1. The synthetic membranes exhibited biological activity at neuroligin-1 densities of approximately 1 to 6 proteins/microm(2). Polyolycarbonate beads with neuroligin-1 covalently attached to the surface failed to activate neurons despite the fact that neuroligin-1 binding activity is preserved. This implies that a lipid membrane environment is likely to be essential for neuroligin-1 activity. This technique allows the study of isolated proteins in an environment that has physical properties resembling those of a cell surface; proteins can diffuse freely within the membrane, retain their in vivo orientations, and are in a nondenatured state. In addition, the synthetic membrane environment affords control over both lipid and protein composition. This technology is easily implemented and can be applied to a wide variety of cellular studies.

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Figures

Figure 1
Figure 1
(A) Schematic diagram of the experiment: GPI-linked neuroligin-1 (red) incorporated into a synthetic lipid bilayer on a silica microbead binds to β-neurexin (green) expressed in a neuron. Formation of the neuroligin–neurexin complex results in the eventual recruitment of scaffolding proteins (grey) and exocytosis-competent synaptic vesicles (blue). (B) Membrane-coated beads were added to hippocampal cell cultures, allowed to settle by gravity, and incubated for 24 h. Transmission image of beads coated with synthetic lipid bilayers containing GPI-linked neuroligin-1, interacting with a hippocampal neuron in culture.
Figure 2
Figure 2
(A) Silver-stained gel of reconstituted and repurified GPI-linked neuroligin-1. Purified, recombinant GPI-linked neuroligin-1 reconstituted into proteoliposomes and subsequently repurified from them runs as a single protein band at the predicted size. (B) FRAP figure demonstrating the mobility of a neuroligin-1 containing lipid bilayer on a 5 μm diameter silica bead. Red fluorescence is due to Texas red-DPPE in the membrane: full illumination prior to bleach (left), exposure pattern during bleach (middle), full illumination 1 min after bleach (right). (C) Application of an electric field demonstrating the mobility of a lipid bilayer containing neuroligin-1 on a planar silica substrate. The strength of the electric field applied to the membrane is 18 V/cm, and the direction of the field is indicated by the arrow on the right: membrane under no electric field (left), electric field applied to membrane after 5 min (center), electric field applied to membrane after 10 min (right).
Figure 3
Figure 3
Presynaptic differentiation triggered by neuroligin-1 on the surface of cells or on lipid bilayer-coated membranes. (A) PC12 cells transfected with neuroligin-1–IRES–GFP (pseudo-colored red for consistency with other panels) were added to a 12 day old hippocampal culture, incubated overnight, then fixed and stained with antisynapsin (green in all panels) to mark presynaptic synaptic vesicles in the axons of the neuron. Synaptic vesicles accumulate, in puncta, at the site of contact with the PC12 cell. (B–F) Twelve day old hippocampal cultures interacting with beads coated with (B) synthetic lipid bilayers containing GPI-linked neuroligin-1 (neuroligin-1 stained in red), (C) synthetic lipid bilayers with no added protein (plain membranes in red), (D) synthetic lipid bilayers containing GPI-linked alkaline phosphatase (GPI-linked alkaline phosphatase in red), (E) neither synthetic lipid bilayer nor protein, bare silica beads (middle panel is DIC image of beads and neurons), and (F) with chemically coupled neuroligin-1 on polystyrene beads (neuroligin-1 in red). Cultures were fixed 24 h following introduction of beads and were stained with anti-HA antibodies for either neuroligin-1 (red), GPI-linked alkaline phosphatase (red), or synapsin (green). Microbeads coated only with synthetic lipid bilayers included 1% Texas red–DPPE in the bilayer for fluorescence imaging. Only beads with synthetic lipid bilayers containing GPI-linked neuroligin-1 induced presynaptic differentiation of the neurons, as indicated by synapsin accumulation in axons contacting the beads.
Figure 4
Figure 4
Adherence of HEK293 cells to polystyrene beads covered with chemically coupled neuroligin-1. White circles denote the positions of polystyrene beads in fluorescence images. (A, B) HEK293 cells not transfected with β-neurexin; no beads are observed to adhere to the cells and have fallen away from the coverslip after inversion of the chamber. (C) Bright field image of polystyrene beads, coupled to neuroligin-1, adhering to HEK293 cells expressing β-neurexin. In all cases, both transfected and nontransfected cells remained adhered to the glass coverslip. (D) Fluorescence image of polystyrene beads, coupled to neuroligin-1, and adhering to β-neurexin-expressing HEK293 cells.
Figure 5
Figure 5
Reference curve of the intensity of emitted light at a wavelength of 522 nm for different concentrations of FITC HA antibody in a 1× PBS solution with 2 vol % Triton X-100 (squares). The plain line is a linear fit to the data. Dashed lines indicate the intensity values measured for the antibody bound to neuroligin-1 incorporated in the synthetic membrane. Three samples were prepared using three different preparations of neuroligin-1, and the range of corresponding concentration of bound antibody is assumed to be directly proportional to the range of neuroligin-1 present in the synthetic membrane.
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