Extended Data Fig. 2: Biochemical and electron microscopy confirmation of the formation of kinesin filaments.
From: A printable active network actuator built from an engineered biomolecular motor
a-b, Biochemical confirmation of CaMLMM filament formation. The solubility of CaMLMM and myosin II in various NaCl concentrations was measured by sedimentation assays and SDS-PAGE. Representative images of n = 2. Uncropped images with molecular weight marker are provided as Source Data. Both CaMLMM and myosin II were found in soluble fractions only at high NaCl concentrations (200 mM or more). Since myosin II is known to form filaments and precipitate in low ionic strength conditions, this result indicates that CaMLMM behaves similarly to myosin. c, Biochemical confirmation of the binding of K465m13 to the CaMLMM filament under the presence of calcium ions. Co-sedimentation of K465m13 was analysed by SDS-PAGE. In the presence of calcium ions, 77% of K465m13 co-sediments with CaMLMM, while most of the K465m13 is found in the supernatant in samples containing EGTA, a calcium chelating agent. This result indicates that K465m13 is bound to CaMLMM in the presence of calcium as designed. d, TEM image of CaMLMM and kinesin filaments (also see Supplementary Discussion 1). Filamentous objects possessing lengths around 0.8 µm, comparable with synthetic myosin filaments, were observed (upper and lower panels). In K465m13-positive samples, the filamentous objects became slightly thicker than the negative control (lower). Close examination of the CaMLMM filaments with K465m13 revealed globular objects attached to the filaments at intervals of around 40 nm (inset), close to that of myosin heads along myosin filaments28, indicating that the arrangement of the K465m13 fusion protein on the kinesin filaments is similar to that of myosin heads in myosin filaments. Representative images of n = 3. Scale bar: 500 nm.
