Microtubule will be polymerized from tubulin monomer using IR light irradiation in a confined cell with a temperature gradient. Under this temperature gradient, the nucleus is formed at the warm end, and the growth process progresses toward the cold end. When the density or length of the filaments exceeds a critical value, well-oriented microtubules with preferential polarity will be obtained (Figure1).
To efficiently create a temperature gradient by IR irradiation, we fix some black tape onto the flow cell. The black color absorbs maximum heat. The region with black tape becomes warm due to IR irradiation, and heat propagates from the warm end to the cold end. Thus, we create the temperature gradient using black tape.
(Figure1: Formation of well-oriented microtubules with preferential polarity polymerized under temperature gradient.)
The well-oriented microtubules will start to move after adding multimeric kinesin and ATP(Figure2).
To produce the artificial sarcomere, we prepare the oriented microtubules with preferential polarity and the multimeric kinesin (the multimeric kinesin is a unit consisting of kinesins assembled by crosslinking of the non-motor domain of kinesin). The minus ends of microtubules grown in the opposite direction are concentrated around the nucleation cite. By adding the multimeric kinesin and ATP to the nucleation cite, the system can undergo contraction.
Kinesins have the tendency to move from the minus to the plus end along the microtubule track. In the figure shown, the upper kinesins will move to the left, whereas the lower kinesins will move to the right.In other words, the upper and lower kinesins tend to move in opposite directions due to anti- parallel microtubules at the nucleation site.However, the multimeric kinesin does not change position because the kinesins are linked strongly.Nonetheless, the kinesins still try to move in the positive direction. As they approach the plus end of microtubule, they move the microtubules instead. In this way, the unit undergoes contraction. This is the artificial sarcomere.
(Figure2: Uniaxial contraction of well-oriented microtubules by adding multimeric kinesin at the nucleation site of microtubules.)
We can form multiple microtubule nucleation sites with temperature gradient created by photo irradiation. After adding kinesin and ATP, it will work as artificial sarcomere(Figure3).
(Figure 3: Schematic diagram of the microtubule nucleation site by light irradiation and an artificial muscle having sarcomere.)