An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

Abstract

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.

Figure 1

Structure of Sib proteins. (A) Organization of the SIBA gene. Exons are shown as numbered black boxes (I–IV). The position of the vector insertion site in sibA mutant cells is indicated (nucleotide 1235 of the coding sequence). Scale bar, 1 kb. (B) A 6-kb SIBA transcript was detected by northern blot in wild-type (WT) but not in sibA mutant cells. (C) Structure of the SibA protein, and of human integrin β. CRD, cystein-rich domain; D.d., Dictyostelium discoideum; H.s., Homo sapiens; IGL, immunoglobulin-like fold; R, repeated motif; SS, cleavable signal sequence; VWA, von Willebrand factor type A domain. (D) The cytoplasmic domain of Sib proteins contains a conserved motif also found in integrin β chains. (E) The transmembrane domain of Sib proteins contains a GxxxG motif also present in integrin β chains.

Figure 2

Interaction of SibA with talin. (A) SibA is localized at the cell surface. The cell surface of wild-type (WT) and sibA cells was biotinylated. Biotinylated SibA was immunoprecipitated and then observed with avidin–horseradish peroxidase. (B) Fusion proteins of glutathione S-transferase (GST) with the cytosolic domain of each Sib protein were produced in bacteria, purified and immobilized on beads. The beads were incubated with Dictyostelium cell lysates, washed and the bound talin was shown by immunoblotting. A lysate from talin mutant cells was used as a control (third lane). The Dictyostelium lysate loaded on the gel (right lane) corresponds to 10% of the amount of lysate incubated with each GST fusion protein. (C) Mutating the membrane-proximal NPxY SibA motif (mut1; see Fig 1D) abolished the interaction with talin.

Figure 3

sibA mutant cells show specific adhesion defects. (A) sibA mutant cells phagocytose latex beads poorly. Wild-type (WT, black curve) or sibA cells (grey curve) were incubated for 20 min in the presence (or absence: white curve) of fluorescent latex beads. The amount of internalized fluorescence was determined with a fluorescence-activated cell sorter. The average number of beads incorporated per cell is indicated above each curve. (B) Cells were incubated with fluorescent latex beads, Klebsiella bacteria (K.p.) or dextran for 20 min. The internalization in sibA cells is expressed as a percentage of WT internalization. The mean and s.e.m. of at least five experiments are indicated. (C) The attachment of sibA mutant cells to a glass substrate is defective. WT and sibA cells attached to glass coverslips were observed by phase-contrast microscopy (Phase) and by IRM (interference reflection microscopy). (D) The areas of 100 individual cells in contact with the coverslip were measured. (E) The actin cytoskeleton is unaffected in sibA cells. WT, sibA or phg2 cells were fixed and stained with phalloidin. As described (Gebbie et al, 2004), abnormal actin aggregates form in phg2 mutant cells. Scale bar, 10 μm.

Figure 4

Development of sibA mutant cells is normal. Wild-type (WT) or sibA cells were starved to induce development. Cellular aggregates were observed after 6 h and fruiting bodies after 24 h. Scale bar, 0.5 mm.