The ampA gene is critical for Dictyostelium discoideum cell migration. To help understand the elusive ampA migrational pathway, second site suppressors were created by REMI mutagenesis. Three novel genes were identified as suppressors of the AmpA overexpressing increased migration phenotype. In order to understand and characterize the novel suppressor gene functions, mRFP fusion proteins were created and knockout cell lines were established. Isolation and characterization results of the three suppressors are described.

REMI mutagenesis was used to identify second site suppressors of a primary mutation in AmpA overexpression. REMI involves the random insertion of a blasticidin resistance cassette into the genome and disruption of residing genes. Disruption of a gene participating in the ampA pathway will produce an alteration in phenotype. I screened for mutants which suppressed the large plaque phenotype in ampAOE cells; the plaque sizes are a reflection of their increased migration. Three suppressors were isolated: lmbd2B, ndm and sma.

Lmbd2b belongs to the evolutionarily conserved LMBR1 protein family, some of whose known members are transmembrane endocytic receptors. Our findings suggest LMBD2B likely functions as a plasma membrane endocytic receptor. Defects in migration were observed in Lmbd2B- cells, they appear to extend excessive pseudopods and have difficulty establishing a front/back orientation to facilitate migration.

The second suppressor is ndm, an F-BAR domain containing protein. It inhibits macropinocytosis, and the increase in endocytosis exhibited by null cells interferes with migration. An interaction between NDM and coronin was found, suggesting it has a role in regulating coronin mediated processes such as macropinocytosis and lamellipodia formation.

The third suppressor, sma, contains a DNA binding SAP domain. It was found to influence many cellular processes similarly to ampA. It was also determined to influence ampA transcript levels. Sma null cells display increased efficiency in chemotaxis, suggesting a critical role for SMA in regulating cell movement. An interesting interaction was found between ampA and sma pathways during chemotaxis, where the presence of AmpA had profound effects on cell movement.

The characterization of the suppressor genes led to the identification of novel genes involved in chemotaxis signaling pathways.