Visualization of the movement of single histidine kinase molecules in live Caulobacter cells

Abstract

The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.

Fig. 1.

Caulobacter crescentus cell cycle. The motile SW cell has a polar flagellum (wavy line) and pili (straight lines) and does not replicate its DNA (nonreplicating DNA represented as a ring). The PleC protein (gray dot) is localized to the flagellar pole of the SW cell. During differentiation into a ST cell, the flagellum is shed and a stalk is built at the same pole, DNA replication initiates (θ structure), and localized PleC is no longer visible. After the ST cell develops into a PD cell, PleC localizes to the new flagellar pole. The PD cell divides to yield a SW cell and a ST cell.

Fig. 2.

Fluorescence images of three strains of Caulobacter and EYFP in a gel. All images are scaled by the intensity of their illumination and are displayed on an identical grey scale. (A) Strain EJ148, expressing PleC-EYFP. (B) Strain EJ153, expressing soluble, cytoplasmic EYFP. (C) Strain CB15N, wild-type Caulobacter. (D) EYFP molecules immobilized in an agarose gel. (Scale bar: 1 μm.)

Fig. 3.

Sequences of fluorescence images, spaced by three different time intervals. The leftmost image in each row is a DF image, to illustrate the orientation of the cell. The second column contains processed DF images for cells in which tracking occurred, showing the position of the cells (gray) and the computed cell midlines (white). (A) Images acquired every 100 ms; note the photobleaching of the lower molecule in the second to last frame. (B and C) Images acquired every 1s. (D) Images acquired every 15 s. The exposure time per image in all cases is 100 ms. (Scale bar: 1 μm.)

Fig. 4.

MSD versus time lag for both the raw data and the data corrected for the 2D projection. Triangles represent the raw planar MSD calculated directly from trajectories. Circles denote the corresponding geometry-corrected true MSD. The fit to the corrected data has a slope of 0.049 μm²/s, yielding a 2D diffusion coefficient D of 12 ± 2 × 10^–3 μm²/s.

Fig. 5.

Percentage of steps in the –s direction. Each bin represents one tenth of the cell, divided lengthwise. In cells with localized PleC-EYFP, bin 1 includes the flagellar pole. Motion in the –s direction is motion toward this pole. In cells without PleC-EYFP localization, one end is arbitrarily assigned as the first bin. Data are not shown for bins 1 or 10 because the s axis arc is not well aligned with the cell axis in these bins.