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. 2012 Nov 5;109(49):E3340–E3349. doi: 10.1073/pnas.1208618109

Fig. 8.

Fig. 8.

Discrimination between MDA5 and MDA5h in filament formation. (A) Fluorescence images of filaments formed by a mixture of MDA5hSNAP (labeled with Alexa Flour 546) and MDA5 (labeled with Hylite647) on 1,012-bp dsRNA. A 1:1 mixture of MDA5hSNAP and MDA5 (100 nM each) was incubated with 1,012-bp dsRNA (0.1 nM), and the complex was immobilized on the flow cell surface. Two-color imaging identified 462 filaments with Alexa 546 fluorescence (Left) and 362 filaments with Hylite 647 fluorescence (Right). Among these, 128 filaments showed common, overlapping positions (such as the one in yellow circle), indicating that these 128 filaments contain both MDA5 and MDA5hSNAP. (B) Venn diagram summarizing the results in A and plot of Alexa 546 vs. Hylite 647 fluorescence intensities of the 128 filaments containing both MDA5 and MDA5hSNAP. (C) Representative traces of MDA5 filament dynamics in the mixture of MDA5 and MDA5hSNAP. Preformed filaments of Hylite 647-labeled MDA5 on 512-bp dsRNA were immobilized on the flow cell surface, and unbound filaments were washed out. A 1:1 mixture of Hylite 647-labeled MDA5 and Alexa 546-labeled MDA5hSNAP (100 nM each) was injected into the flow cell with ATP or ADPCP (2 mM). The fluorescence intensities of MDA5 and MDA5hSNAP were normalized against those of the filaments formed by the respective protein alone. (D) Averaged traces of MDA5 filament dynamics in the mixture of MDA5 and MDA5hSNAP. Experiments were performed as in C. n = 325 and 396 for ATP and ADPCP reactions, respectively. (E) Proposed model for length discrimination by MDA5. With ATP, MDA5 filaments continuously switch between assembly and disassembly phases. Because disassembly occurs primarily from filament ends, filaments on short dsRNA more frequently undergo a complete disassembly, which requires slow de novo nucleation for rebinding. In contrast, filaments on long dsRNA alternate between partial disassembly and fast elongation, bypassing nucleation. Thus the slow nucleation kinetics amplifies the time constant (τ) in Fig. 6B.