Chemotaxis of <em>Dictyostelium discoideum:</em> Collective Oscillation of Cellular Contacts

<div><p>Chemotactic responses of <em>Dictyostelium discoideum</em> cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single <em>Dictyostelium discoideum</em> cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of <em>Dictyostelium discoideum</em> cells.</p> </div

Impedance signal of <i>D. discoideum</i>.

<p>Magnitude of detrended impedance signal at 4 kHz |<i>Z</i>_Detrend|_{4 kHz} of <i>D. discoideum</i> (3,750 cells mm⁻² added in Sorenseńs buffer) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054172#pone-0054172-g001" target="_blank">Figure 1</a> C. Cells were seeded at <i>t</i> = 0 min on a circular gold electrode ( = 250 µm). Boxes highlight magnification of the impedance signal. Data were smoothed by subtracting a moving average algorithm (box size: 800 points) to remove long-term trends.</p

Changes in circularity of <i>D. discoideum</i>.

<p>A) Optical micrographs of the ameba-covered electrode taken at two different times as marked by the dashed line in (B). Cells’ perimeters were manually surrounded by a green line to compute circularity. For sake of clarity, the electrode is eradicated from the bottom part of the images. B) Circularity <<i>C</i>> of <i>D. discoideum</i> cells determined as a function of time (red dots). Additionally, the corresponding time series of the detrended impedance values |<i>Z</i>_Detrend|_{4 kHz} from the same area is shown in blue.</p

D-QCM of <i>D. discoideum</i> chemotaxis.

<p>D-QCM measurement of starved <i>D. discoideum</i> amebas. Shift in resonance frequency (red) and damping (black) of an oscillating quartz crystal as a function of time. <i>D. discoideum</i> cells (10,000 cells mm⁻²) were seeded at <i>t</i> = 0 on a gold-electrode. The black box highlights the time period during which collective oscillations occur.</p

Periodic clustering of <i>D. discoideum</i>.

<p>A) Optical micrographs (bright field images) of the cell-covered electrode at <i>t_I</i> = 387.2 min and <i>t</i>_II = 390.2 min after seeding of <i>D. discoideum</i> cells (3750 cells mm⁻²). The cells marked in green are isolated amebas, while blue color indicates cells belonging to a 2-D aggregate or cluster. B) Time series of the number of isolated cells derived from image analysis (red curve). Time points labeled with gray lines correspond to the images shown in (A). Additionally, the corresponding time series of the simultaneously acquired detrended impedance values |<i>Z</i>_Detrend| are shown as a blue line.</p

TIRF analysis of <i>D. discoideum</i> chemotaxis.

<p>A) Bright field and B) TIRF microscopy image sections of cells (8,000 cells mm^-²) starved for 5 h on a glass substrate recorded at three distinct time points (I, II, III). The bright field images are recorded 10 second earlier than the TIRF images presented beneath. The arrow exemplarily highlights one particular cell during the stages I-III. C) Plot of the measured total fluorescence intensity of the whole TIRF image as a function of time shows an oscillation, which is cropped and magnified in the second plot. The first image (I) in B corresponds to a peak minimum of the fluorescence intensity and the third image (III) to a peak maximum.</p

Cross correlation coefficients.

<p>Cross-correlation coefficient <i>ρ</i> of the corresponding descriptors, cell number <i>N</i>, mean covered area per cell <i>A</i>, mean circularity <<i>C</i>>, TIRF intensity (time axis multiplied with 1.94 to match periodicity of impedance spikes), and number of isolated cells <i>N</i>_s with impedance time traces. Amebas were displaying oscillations in impedance recorded at 4 kHz generator frequency.</p

Origin of impedance oscillation

<p>. The scheme illustrates how circularity , number of isolated amebas no., light intensity of subtracted bright field BF, and fluorescence intensity from TIRF mages (TIRF) correspond temporally to the measured impedance spikes |<i>Z</i>|.</p

Cell number and covered area of the electrode.

<p>A) Temporal evolution of the number of <i>D. discoideum</i> cells on the electrode. B) Ameba-covered area of the ECIS-electrode obtained from bright field microscopy (red). Simultaneously recorded impedance |<i>Z</i>_Detrend|_{4 kHz} (blue).</p

Correlation between TIRF and ECIS experiment.

<p>A) Oscillating gray tone of subtracted bright field images (red) in comparison to the total fluorescence intensity of the corresponding TIRF images (green) and the impedance signal (black) as a function of time. The procedure allows to correlate the two independent experiments. B) Scheme of the two proposed states that amebas assume during TIRF microscopy explaining low fluorescence intensity (left) and high intensity (right). The minimal cell-substrate distance is not significantly undercut, only the overall contact zone increases leading to larger fluorescence intensity and impedance.</p