Talin-Null Cells of Dictyostelium Are Strongly Defective in Adhesion to Particle and Substrate Surfaces and Slightly Impaired in Cytokinesis

Abstract

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Phg2, a Kinase Involved in Adhesion and Focal Site Modeling inDictyostelium

“…The phg1 mutant was described previously (Cornillon et al, 2000) The myoVII and talin knockout mutants were derived from DH1-10 by using the knockout plasmids described previously (Niewohner et al, 1997;Titus, 1999). The phg2 mutant strain was isolated following an identical procedure to that used to isolate the phg1 mutant (Cornillon et al, 2000).…”

“…As detailed above, several other gene products also play a critical role in cellular adhesion in Dictyostelium, in particular myosin VII and talin (Titus, 1999;Niewohner et al, 1997;Tuxworth et al, 2001). To compare their role in adhesion with the function of Phg2, we generated the corresponding disruption mutants in our Dictyostelium laboratory strain (DH1-10), and measured the adhesion of phg1, phg2, myoVII, and talin cells to a glass substrate in HL5 medium.…”

“…Previous reports have shown that myosin VII and talin, both actin-binding proteins, are involved in the organization of the actin cytoskeleton (Niewohner et al, 1997; The Phg2 kinase domain fused to GST was produced in bacteria, partially purified, and used to phosphorylate in vitro a bacterial protein in the presence of [␥-32 P]ATP. The phosphorylated protein was purified and hydrolyzed, and the phosphorylated amino acid residues were separated by electrophoresis in the first dimension and ascending chromatography in the second dimension.…”

“…Based on the analysis of specific knockout mutants, several orthologues of mammalian adhesion proteins were shown to play essential roles in cellsubstrate adhesion in Dictyostelium (Bracco et al, 2000). For example, the actin-binding protein talin is necessary for phagocytosis and adhesion in Dictyostelium (Niewohner et al, 1997). Dictyostelium myosin VII null mutants also have defects in both cell-cell and cell-substrate adhesion (Tuxworth et al, 2001).…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…A subset of actin-binding proteins associate transiently with the phagocytic cup of amoebae such as talin (Niewohner et al 1997) and coronin, which is thought to regulate the G-to F-actin equilibrium (Maniak et al 1995). Furthermore, macropinocytosis and phagocytosis share many components of the cytoskeletal machinery involved in formation of membrane ruffles, but their trigger and regulation are probably different (Maniak 1999).…”