Modeling a Microtubule Filaments Mesh Structure from Confocal Microscopy Imaging

Abstract

This study introduces a modeling method for a supermolecular structure of microtubules for the development of a force generation material using motor proteins. 3D imaging by confocal laser scanning microscopy (CLSM) was used to obtain 3D volume density data. The density data were then interpreted by a set of cylinders with the general-purpose 3D modeling software Blender, and a 3D network structure of microtubules was constructed. Although motor proteins were not visualized experimentally, they were introduced into the model to simulate pulling of the microtubules toward each other to yield shrinking of the network, resulting in contraction of the artificial muscle. From the successful force generation simulation of the obtained model structure of artificial muscle, the modeling method introduced here could be useful in various studies for potential improvements of this contractile molecular system.

Keywords: artificial muscle; fluorescent microscopy; kinesin; microtubule; molecular machine; molecular robotics.

Figure 1

Imaging results of the network structure of microtubules in an artificial muscle specimen, by confocal laser scanning microscopy (CLSM): (a) the microtubule concentration at 10 μM, (b) the concentration at 0.1 μM. The inset box in (b) was used for the 3D volume data construction in this study, and (c) an assembly of microtubules without kinesin linkers. Microtubule filaments labeled with Alexa488 dye visible in green color, while the DNA origami binding region is labeled with TAMRA dye and is visible in red color. Most filament bodies of microtubules bind both dyes and present a yellow appearance. Scale bars, 10 μm.

Figure 2

Schematic diagram of two microtubule aster structures for the contraction system. The aster structure was mediated by a DNA origami linker, indicated by red circles. The multimeric kinesin linker binds two microtubule filaments between asters to pull the asters closer, depicted as two dumbbells. Aster fragments are depicted as solid line filaments, omitting the dashed line filaments. A gray filament on the left side is an example of the dissociation that may occur to fragment the aster structure.

Figure 3

A volume rendering for the obtained 3D depth slice image dataset by CLSM. Microtubule filaments stacks were inclined toward each other. Background noise was visually decreased by low signal level cut off. The white rectangle box is 35 × 35 × 14 μm.

Figure 4

(a) The surface representation for obtained 3D volume data by z-slice images. Two isosurface values at high density (brown) and low density (blue) are depicted. Some portions of the blurred volumes showed less intensity. (b) Cylinder models of microtubules fitted to the observed volume data. We also tried to fit the direction of the cylinders, with one end marked in red, to the observation shown in Figure 3. Most network structures were well described by these cylinders, however, at regions where the cylinders intersect, it was difficult to recognize their geometrical components. The center lines for each cylinder are also depicted to indicate the real microtubule filament diameter of 25 nm. This inflation of the cylinder radius due to the resolution limit is discussed in Section 4.1.

Figure 5

The motility element in the microtubule filaments network: the motion of multimeric kinesin linker on the two microtubule filaments at the three states of a possible contraction motion are depicted for (a) starting, (b) running, and (c) ending. The tentative model with three sets of fragments of microtubule asters. Applying motion to those three states of motion yields the shrinking of the network: (d) starting, (e) running, and (f) ending.

Figure 6

(a) A predicted filament structure model for smooth muscle. Cyan ellipsoids designate the dense body that binds actin filaments depicted in red. Actin filaments were bundled to form a broom-like structure. Two of these structures will hold a myosin filament with protruding myosin heads. A motor protein is depicted in blue. (b) A magnified view of the actin and myosin filaments interacting on the overlapped regions.