Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo

Abstract

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Keywords: Notch signaling; low tension gauge tether (LTGT); nano yoyo; single-molecular forces.

Figure 1

Design and working principle of DLL1-LTGT. (a) Force induced activation of Notch receptors upon binding to ligand DLL1 is shown. (b) Ligand DLL1 is conjugated to a double stranded DNA with an overhang of ssDNA (dT₆₅) wrapped around a homotetrameric single-tailed SSB. DLL1-LTGT was immobilized to the passivated glass surface via biotin-neutravidin interactions. Glass surfaces were also coated with fibronectin to promote cell adhesion. A Cy3 fluorophore is conjugated to DLL1-LTGT so that we can monitor fluorescence signal loss in real time when a cell pull away the construct.

Figure 2

Force calibration of btSSB: ssDNA LTGT. (a) High-resolution optical tweezers were used to determine required force for dissociation of ssDNA from btSSB. (b) A histogram of dissociation force between a single ssDNA and a single SSB is shown here (n=47). A Gaussian fit to the distribution gives a mean dissociation force of 4.1±0.1pN and a FWHM of 3.2±0.3 pN.

Figure 3

Notch signaling in transgenic CHO-K1 cells is not activated in DLL1-LTGT assay as represented by low H2B-YFP expression. (a–b) High levels of YFP signal indicate Notch signaling is activated when ligand DLL1-Fc (10 nM) is directly immobilized on the surface. (c–d) When ligand DLL1 is excluded, Notch signaling is not activated. (e–f) Notch signaling is also not activated on the DLL1-LTGT surface even with ten-fold elevated concentration (100 nM). (g–h) Cells on 12 pN and 54 pN TGT surfaces show activation of Notch signaling. When btSSB: ssDNA part is excluded from the construct, one may design TGT in both 12 pN and 54 pN orientation simply changing the biotin position. It is to be noted that cells can activate Notch signaling even on 12 pN TGT engineered surfaces. This suggests Notch activation requires force between 4–12 pN.

Figure 4

Time dependent rupture of DLL1-LTGT from the surface. DIC, surface fluorescence, and analyzed images show very little rupture after 1 hour of cell plating. However, there is a significant difference observed in terms of LTGT rupture after 2 hours suggesting that Notch receptors can dissociate ssDNA tethers from surface immobilized btSSB. Red, green and blue regions indicate background, rupture region, and cell nuclei respectively. The baseline value was obtained from non-fluorescent images and were corrected from both ruptured and background regions. A histogram of each region was plotted and the fit to the histogram was used to calculate rupture percentage. Values next to each peak indicate mean intensity of each region.