Figure 1. A tension sensor in mini-Nesprin-2 is under actin-dependent tension when mouse keratinocytes (MKCs) engage the extracellular matrix through integrins.

(A) Cartoon of the N2G-JM-TSMod, in which the mTFP-Venus tension sensor module is inserted between the cytoplasmic domain and the C-terminal KASH domain/transmembrane domain of mini-Nesprin-2. Tension leads to a reduction in the fluorescence resonance energy transfer (FRET) index. Cartoon of the NoT_TSMod control, in which the TSMod resides at the N-terminus of the N2G, and therefore cannot experience intramolecular tension. (B, C) The N2G-JM-TSMod displays higher tension (lower FRET index) than the no-tension NoT_TSMod control (higher FRET index) when expressed in MKCs plated on FN-coated glass. Representative images show that, at low expression levels, both the N2G-JM-TSMod and NoT_TSMod are successfully targeted to the nuclear envelope. Images are pseudocolored according to the normalized FRET index (Feige et al., 2005). The median FRET index value of cells expressing the NoT_TSMod was set to a value of 1 in (C) and used to scale to relative values for the N2G-JM-TSMod. (D, E) Disruption of actin filaments with 0.5 μM latrunculin A (Lat A) decreases the tension on the N2G-JM-TSMod, leading to a higher FRET index. The median FRET index of cells expressing the N2G-JM-TSMod was set to a value of 1 in (E). (F, G) Tension on the N2G-JM-TSMod is sensitive to substrate mechanics. Plating of MKCs on FN-coated compliant substrates (3 kPa PDMS) leads to increased FRET compared to MKCs plated on FN-coated glass. (H, I) Plating of MKCs on extracellular matrix that engages integrins drives high tension on the N2G-JM-TSMod. MKCs grown on glass coated with fibronectin or laminin drive a higher tension state (low FRET index) than for cells grown on glass coated with poly-L-lysine (high FRET index). The median FRET index value of cells expressing the N2G-JM-TSMod plated on FN-coated glass was set to a value of 1 in (G) and (I). Data for FN replotted from (I). (J, K) MKCs lacking β1 integrin fail to drive high tension on the N2G-JM-TSMod. Representative images demonstrate that the N2G-JM-TSMod is successfully targeted to the nuclear envelope in wild-type (WT) and β1 integrin null MKCs. Higher FRET indexes at the nuclear envelope in β1 integrin null MKCs demonstrate that β1 integrin engagement with the extracellular matrix is required for high tension on the N2G-JM-TSMod. The median FRET index value of cells expressing the N2G-JM-TSMod plated on fibronectin-coated glass was set to a value of 1 in (K). For all plots, errors reflect SD, n ≥ 30 cells for each condition measured from n = 3 experiments. ****p<0.0001 as determined by unpaired t-test (C, E, G, K) or one-way ANOVA (I). All scale bars = 5 μm.

Figure 1—figure supplement 1. Validation controls for the N2G-JM-TSMod.

(A, C, D) Representative images of the ‘dark’ control constructs (mTFP donor or Venus acceptor fluorescence only) used for bleed through assessment as required for fluorescence resonance energy transfer (FRET) experiments. For comparison, representative images of the N2G-JM-TSMod construct in which fluorescent signals can be readily detected in the donor, acceptor, and FRET channels are included. (B) The FRET index for the NoT_TSMod control is unaffected by actin depolymerization. In wiild-type mouse keratinocytes expressing the NoT_TSMod control, there is no significant (ns) change in FRET index as assessed by unpaired t-test upon addition of 0.5 μM latrunculin A (Lat A) compared to vehicle controls. Data for vehicle alone replotted from Figure 1C. Errors reflect SD, n ≥ 30 cells for each condition measured from n = 3 experiments. Scale bar = 5 μm.