Cell Substratum Adhesion during Early Development of <i>Dictyostelium discoideum</i>

<div><p>Vegetative and developed amoebae of <i>Dictyostelium discoideum</i> gain traction and move rapidly on a wide range of substrata without forming focal adhesions. We used two independent assays to quantify cell-substrate adhesion in mutants and in wild-type cells as a function of development. Using a microfluidic device that generates a range of hydrodynamic shear stress, we found that substratum adhesion decreases at least 10 fold during the first 6 hr of development of wild type cells. This result was confirmed using a single-cell assay in which cells were attached to the cantilever of an atomic force probe and allowed to adhere to untreated glass surfaces before being retracted. Both of these assays showed that the decrease in substratum adhesion was dependent on the cAMP receptor CAR1 which triggers development. Vegetative cells missing talin as the result of a mutation in <i>talA</i> exhibited slightly reduced adhesive properties compared to vegetative wild-type cells. In sharp contrast to wild-type cells, however, these <i>talA</i> mutant cells did not show further reduction of adhesion during development such that after 5 hr of development they were significantly more adhesive than developed wild type cells. In addition, both assays showed that substrate adhesion was reduced in 0 hr cells when the actin cytoskeleton was disrupted by latrunculin. Consistent with previous observations, substrate adhesion was also reduced in 0 hr cells lacking the membrane proteins SadA or SibA as the result of mutations in <i>sadA</i> or <i>sibA</i>. However, there was no difference in the adhesion properties between wild type AX3 cells and these mutant cells after 6 hr of development, suggesting that neither SibA nor SadA play an essential role in substratum adhesion during aggregation. Our results provide a quantitative framework for further studies of cell substratum adhesion in <i>Dictyostelium</i>.</p></div

Substratum adhesion in cells lacking <i>sibA</i> or s<i>adA</i>.

<p><b>A</b> Cell-substratum adhesion of wild type Ax3, <i>sibA</i> and <i>sadA</i> null cells at either 0 or 5 hr of development as measured by the microfluidic assay from the remaining fraction of cells after 40 minutes. <b>B</b> Work of adhesion W_Adh. of wild type (AX3) and cells lacking either <i>sibA</i> or <i>sadA</i> was measured after 0 hr (<i>n</i> = 29 and 41) and 6 hr (<i>n</i> = 45 and 41) of development. Significance was judged from the Wilcoxon-rank-sum test.</p

Developmental regulation of the decrease in adhesion.

<p><b>A</b> Wild type (AX3) and <i>carA</i>⁻ cells lacking the cAMP receptor after 0 and 5 hr of development were analyzed with the microfluidic assay for the remaining fraction of cells after 40 minutes. <b>B</b> Work of adhesion (W_Adh.) of wild type (AX3), cells lacking the cAMP receptor (<i>carA</i>⁻) or cells lacking talin (<i>talA⁻</i>) was measured after 0 hr (veg <i>n</i> = 36) and 6 hr (dev <i>n</i> = 30 for <i>carA⁻</i>, <i>n</i> = 34 for <i>talA⁻</i>) of development. Nonparametric statistical hypothesis test Wilcoxon rank-sum test was used for significance.</p

Single Cell adhesion Force Spectroscopy assay.

<p><b>A.</b> Force was measured from bending of the cantilever and a typical force-separation curve is shown for an approach-retraction cycle highlighting the two assessed parameters: maximum adhesion force F_Max.Adh. and the work of adhesion W_Adh. (integral of the hatched area). I–IV refers to the four panels shown under B. <b>B.</b> Side view of a cycle of approach and retraction of a cell attached to a cantilever. Panels I and II: A cell can be seen hanging below the cantilever as it approaches the glass slide (light colored surface, <i>Dictyostelium</i> contour in red under I). Panels III and IV: The cell can be seen to remain on the cantilever as it is retracted and it maintains a rounded shape. Arrows highlight cell position. Scale bar is 20 µm.</p

Decrease in adhesion during early development of <i>Dictyostelium discoideum</i>.

<p><b>A.</b> Microfluidic assay: the remaining fraction of cells after 40 minutes in chambers 2–8 is shown for cells that had developed for varying lengths of time. latB refers to 0 hr cells that were treated with 10 µM latrunculin B for 30 minutes to disrupt the actin cytoskeleton and assayed in the presence of the drug. Average of at least 5 independent experiments. The bars indicate the S.E.M. <b>B.</b> Single cell adhesion force assay of cells during early development. Box plot of the distribution of maximum adhesion forces F_Max.Adh of individual single cells, where the bottom and the top of the box represents the first and the third quartiles, and the band corresponds to the median. Cells were developed for 0 h (<i>n</i> = 33), 3 h (<i>n</i> = 47), 6 hr (<i>n</i> = 31). Top whiskers are at 90% and bottom whiskers are at 10% of the distribution. Latrunculin B treated 0 hr cells (Lat) (<i>n</i> = 27) were assayed in the presence of 10 µM latrunculin B to disrupt the actin cytoskeleton. <b>C.</b> Work of adhesion W_Adh. measured for the same cells. Nonparametric statistical hypothesis test Wilcoxon rank-sum test were used for significance.</p

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

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

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

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

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