Analysis of chemotaxis in Dictyostelium

Abstract

Dictyostelium discoideum is an excellent model organism for the study of directed cell migration, since Dictyostelium cells show robust chemotactic responses to the chemoattractant cAMP. Many powerful experimental tools are applicable, including forward and reverse genetics, biochemistry, microscopy, and proteomics. Recent studies have demonstrated that many components involved in chemotaxis are functionally conserved between human neutrophils and Dictyostelium amoebae. In this chapter, we describe how to define the functions of proteins that mediate and regulate cell motility, cell polarity, and directional sensing during chemotaxis in Dictyostelium.

Fig. 1

(a) Development of cells on non-nutrient agar plates. (b) Chemotaxis to a pipette filled with 1 μM cAMP

Fig. 2

G protein activation upon cAMP stimulation monitored by FRET between Gα2-CFP and Gβ-YFP. Loss of FRET (decreased YFP:CFP ratio) was induced when a micropipette containing 10 μM cAMP was introduced at 45 sec and persisted until the micropipette was removed at 180 sec.

Fig. 3

Visualization of transient PIP₃ production upon uniform cAMP stimulation. Cells expressing PHcrac-GFP were examined by fluorescence microscopy (a) and immunoblotting using anti-GFP antibodies (b) after addition of 1 μM cAMP. T, 20% of input.

Fig. 4

PKB activation in response to chemoattractant stimulation. (a) Schematic representation of the activation of PKBR1 and PKBA. The N-ternimus of PKBR1 is myristoylated while the N-terminus of PKBA has a PIP₃-specific PH domain. Upon cAMP stimulation, the two PKBs are activated through phosphorylation of their HMs by TorC2 and ALs by PDK. The phosphorylation sites (P) can be detected by phospho-specific antibodies. (b) Wildtype cells were stimulated with cAMP, lysed at the indicated time points, and subjected immunoblotting using antibodies against phospho HM (top panel), phospho-AL (middle panel), and phospho-substrate (bottom panel).

Fig. 5

Ras activation in response to chemoattractant. (a) cAMP stimulation triggers rapid and transient activation of endogenous Ras proteins, which can be pulled down by GST-RBD-Byr2. (b) Fluorescent images of GFP-RBD-Raf1 in wildtype cells before and after cAMP stimulation.