Journal: bioRxiv

Article Title: The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

doi: 10.1101/2020.05.03.075085

Figure Lengend Snippet: (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-Nesprin2. Tension leads to a reduction in the 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 mouse keratinocytes (MKCs) plated on fibronectin-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 . 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 fibronectin-coated compliant substrates (3 kPa PDMS) leads to increased FRET compared to MKCs plated on fibronectin-coated glass. (H,I) Plating MKCs on extracellular matrix that engages integrins drives high tension on the N2G-JM-TSMod. MKCs grown on glass coated with fibronectin or laminin drives 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 fibronectin-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 WT and β1 integrin KO MKCs. Higher FRET indexes at the nuclear envelope in β1 integrin KO MKCs demonstrates 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.

Article Snippet: WT and Sun dKO keratinocytes were plated in a fibronectin-coated (50ng/ml; Sigma Aldrich) 6-well dish such that they were 70-80% confluent.

Techniques: Expressing