Fig. 4: MDA5–LGP2 microfilaments, but not ATM clusters, mediate downstream signaling. | Cell Research

Fig. 4: MDA5–LGP2 microfilaments, but not ATM clusters, mediate downstream signaling.

From: ATP-dependent one-dimensional movement maintains immune homeostasis by suppressing spontaneous MDA5 filament assembly

Fig. 4

a A schematic illustration of the MAVS-CARD recruitment experiment. b The frequency of MAVS-CARD recruitment by MDA5 and MDA5ΔN under various conditions (mean ± SD; n = number of dsRNA molecules). c Representative kymographs showing the recruitments of AF647-MAVS-CARD (100 nM) by Cy3-MDA5 (20 nM) in the absence or presence of LGP2 (20 nM). Cy3-MDA5 is shown in green and AF647-MAVS-CARD is shown in red. d Representative kymographs showing the recruitments of AF647-MAVS-CARD (100 nM) by Cy3-MDA5ΔN or Cy3-MDA5 (20 nM) in the presence of LGP2 or LGP2(intermut) (20 nM). Cy3-MDA5 is shown in green and AF647-MAVS-CARD is shown in red. e A schematic illustration showing the two-colored single-molecule co-localization assay. f Representative kymographs showing the formation of MDA5-LGP2 microfilaments on a 1000-bp dsRNA substrate using two-colored single-molecule co-localization assay. g Representative trajectories showing the formation of an ATM cluster without LGP2, and the formation of a microfilament with LGP2 on a 1000-bp dsRNA substrate. The dotted line indicates the peak fluorescence intensity. h The distribution of peak fluorescence intensity of MDA5 (15 nM) under various concentrations of LGP2 (0 nM, 3 nM, 5 nM, 10 nM, and 15 nM) on 1000-bp dsRNA substrate (n = MDA5 binding events). i The frequency of MAVS-CARD recruitment by MDA5 (15 nM) and various concentrations of LGP2 (0 nM, 3 nM, 5 nM, 10 nM, and 15 nM) on a 1000-bp dsRNA substrate (mean ± SD; n = number of dsRNA molecules). j Column plots of relative IFNβ mRNA levels showing the MDA5 signaling activity under various conditions. 293∆RIG-I (endogenous RLRs), 293∆RIG-I with MDA5 overexpression (MDA5), and 293∆RIG-I with MDA5 and LGP2 co-overexpression (MDA5+LGP2) were stimulated with dsRNA substrates of different lengths. All single molecule studies were performed at least two separate times. All cell-based assays were repeated independently at three times with similar results. Error bars represent SEM between measurements, centered on the mean of a single experiment.

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