Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

The overall goal of this procedure is to examine the activity of various components of the intracellular signal transduction network following brief mechanical perturbation. This method can help answer key questions in the field of cell migration such as how cells sense and directionally migrate toward diverse environmental cues including mechanical stimuli. The main advantage of this technique is that it allows examination of the initial response to mechanical stimulus without the confounding contribution of motility or polarity to the response.

We first had the idea for this method when we tested the response of adherent cells to chemoattractant which was delivered with a slight agitation and we noticed a robust response of cells stimulated with vehicle control. To begin, prepare Klebsiella aerogenes by inoculating a small number of cells from frozen stock into HL5 medium without antibiotics and incubate the culture overnight. Growth of Dictyostelium in the presence of Klebsiella aerogenes bacteria will reduce the number of macropinosomes which will improve cell responsiveness to external stimuli.