Figure 6.

SPOTs binding to cells expressing sfGFP-gB

(A) In (i) is a confocal slice from live-cell fluorescence microscopy of HEK293T cells expressing sfGFP-tagged gB from HSV-1 (green) ∼15 min after addition of Cy5-labeled SPOTs (blue). SPOTs quickly localize to the surface of cells expressing sfGFP-gB. In (ii), the pattern of SPOT binding is similar to that observed with α-GFP antibodies conjugated to Alexa-647 (red). In (iii), no evidence of cell surface binding is observed when SPOTs are added to cells expressing mCherry-gB (magenta). Scale bars, 5 μm.

(B) Two low-magnification cryoEM projection images of SPOTS bound to the surface of BHK-21 cells expressing sfGFP-gB (i and ii). SPOTs are indicated with blue arrow heads. Scale bar, 250 nm.

(C) Sequential computed tomographic slices of the areas indicated in (Bi) and (Bii), respectively. Although the SPOTs (blue arrow heads) often extend across multiple slices, it is still possible to trace the post structure to the membrane in many cases. Slice thickness is ∼2.8 nm. Scale bars, 50 nm. Average defocus across the tilt series is ∼−3.8 and −4.5 μm for (i) and (ii), respectively.

(D) Manual segmentation of the tomograms in (C). The signpost origami structure (various shades of blue) from Figure 2 is fit into each of the segmented SPOTs (gray mesh). The plasma membrane is shown in green, with protein density on the membrane surface in light green. Scale bars, 50 nm.