Figure 4.

Simultaneous determination of the helicity and rotational direction of archaellum by cross-kymography analysis (a) The schematics of relationships between the direction of rotation (clockwise, CW; counterclockwise, CCW) and helicity (right-handed, RH; left-handed, LH). We first defined the configuration as archaella aligned parallel to the north–south orientation and the cell body located at the north; additionally, the rotation direction is defined as the observer looking at the archaella from the protrusion direction outside the cell body. Note that a camera is set on the left-side camera port, and images are always mirror images because the totally reflecting prism inside the inverted microscope reflects, just once, the light coming through the objective. The portion of filaments that made contacts with the evanescent field was visible as short lines aligned northwest-southeast relative to the major axis of the cell body, indicating the right-handed helicity. On the contrary, the orientation of filaments is northeast-southwest, indicating the left-handed helicity. (b) The schematics to explain how to determine the helicity from the east-west kymograph. Light-blue rectangle represents the location to make the east-west kymograph. Cyan and pink arrow indicated the direction of wave propagation. Gray, orange, magenta and green ellipse represents the time course of wave propagation. Therefore, we could detect the transition of fluorescent signal at the rectangle as shown by orange or green arrows. (c) Left: definition of axes. Right: matrix of combinations to determine the directional rotation and helicity of helical structure from cross-kymography, which is the analysis of two kymographs from two different lines in the same image that perpendicularly align with each other in the region of interest. (d–g) Left: Fluorescent micrograph. Right: two kymographs taken from orthogonally oriented lines on left micrograph. In north-south kymograph, the right-handed archaellar wave propagates away (d) and towards (e) the cell body, which guaranteed the CW and CCW rotation, respectively. On the contrary, the left-handed flagellar wave propagates away (f) and towards (g) the cell body, which guaranteed the CCW and CW rotation, respectively. In the east-west kymograph, the helicity could be uniformly determine as mentioned above. Pseudo-colors of kymographs indicate the direction of spot propagation, which correspond to c. The figure was reused with permission from Kinosita et al., 2016 with modifications.