Cell adhesion in Dictyostelium discoidum

Crawling cells like amoebae, leukocytes, and many tumor cells, attach to diverse substrates as they migrate over long distances or across barriers. Amoebae move rapidly during chemotaxis, leucocytes migrate across barriers to get to their site of action, and tumor cells suddenly detach from their primary site and actively invade other parts of the body. For this rapid low-affinity crawling to occur, substrate adhesion is an important process. Because, in these cells, little is known about the molecules that are involved in substrate adhesion, we are using the single cellular model organism Dictyostelium discoidum to address this question.

The social amoeba Dictyostelium lives in the soil and feeds on bacteria. Upon depletion of the food source, starving amoebae enter a life cycle that culminates in the formation of a multicellular fruiting body. In the early stages of this process, during chemotaxis, the10µm-amoebae migrate up to 20 µm / minute. Because they are very motile these cells cannot afford to form strong interactions with their substrate like focal adhesions or focal attachments. However, as living conditions in the soil can differ enormously, the amoebae attach to virtually any substrate. Dictyostelium is an ideal model system to study adhesion and motility because it is amenable to simple genetics (the cells are haploid), biochemistry (the cells can be grown in large quantities), and immunohistochemistry. The Dictyostelium genome project is also underway. For more information on Dictyostelium visit http://dictybase.org/.

In order to identify molecules, or whole pathways, which are responsible for substrate adhesion in Dictyostelium, we performed a genetic screen using insertional mutagenesis. This is accomplished by restriction enzyme mediated integration (REMI), which involves transformation of the cells with a linearized plasmid, together with the restriction enzyme that was used to cut the plasmid. The enzyme cuts the genomic DNA and thus facilitates the stable plasmid insertion into random genomic sites, often resulting in the disruption of a gene. Transformed cells were selected by drug resistance. Cells that were unable to attach were selected by passing on floating cells only, discarding the attached cells (illustrated in Fig.1). After a few rounds of selection for cells that could not adhere, we isolated several different substrate adhesion deficient mutants (Sad mutants). The mutant cells grow detached from the plastic surface of a petri dish and have a complex phenotype. They are often larger than wildtype (WT), never spread, and have a blebby appearance (Fig. 2 shows a selection of three different mutants).

Fig. 2: Three substrate adhesion mutants. Growing sadA, sadB, and SadC mutant cells, in comparison to wildtype (WT). Mutant cells often have blebs, a wide variety of sizes, and sometimes grow in clumps. Note that some cells are completely out of focus, because they are floating in different focal plains.

We are currently in the process of genetically and phenotypically analyzing these mutants. Most progress has been made with mutant sadA; here we found that the plasmid inserted into a novel adhesion molecule, which we named sadA. Fig. 3 illustrates the sadA locus and plasmid insertion site. A manuscript describing the initial characterization of this mutant is in preparation.

Fig. 3: SadA locus. The sadA gene is approximately 4 kb long and contains 3 exons (dark gray), separated by two short introns (light gray). The plasmid inserted about 900 bp downstream of the translation initiation site, disrupting the translation of the remainder of the gene.