Abstract
Vegetative and developed amoebae of Dictyostelium discoideum 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 talA exhibited slightly reduced adhesive properties compared to vegetative wild-type cells. In sharp contrast to wild-type cells, however, these talA 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 sadA or sibA. 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 Dictyostelium.
Highlights
Motile cells must have traction on the substratum to extend the anterior pseudopod and retract the rear
While much is known about the structure and function of focal adhesion complexes of mammalian cells binding to proteins of the extracellular matrix, it is becoming clear that such specialized complexes are not always necessary for cell substrate adhesion and motility [4,5]
We used the cantilever of an Atomic Force Microscope (AFM) to hold a cell and measure the forces needed to detach it from a glass surface (Fig. 1) (Single Cell adhesion Force Spectroscopy, single cell force spectroscopy (SCFS)) [22,23,24,25,26]
Summary
Motile cells must have traction on the substratum to extend the anterior pseudopod and retract the rear. The cytoplasmic face of heterodimeric integrins associates with various actin-binding proteins, such as talin, a-actinin, paxillin, vinculin or filamin. They tether to the F-actin of the cytoskeleton forming focal adhesions that remain fixed to the matrix until the cell has moved over them. While much is known about the structure and function of focal adhesion complexes of mammalian cells binding to proteins of the extracellular matrix, it is becoming clear that such specialized complexes are not always necessary for cell substrate adhesion and motility [4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
