Phenotypic analysis of two unicellular Gonium pectorale mutants defective in extracellular matrix assembly

Abstract

The evolution of multicellularity is a major transition in the morphological organization of organisms, however, the molecular mechanisms important for this transition in any taxa are currently not well understood. In most taxa, the molecular signature of the transition to multicellularity is obscured by nearly a billion years of divergence. In contrast, multicellularity evolved recently in the volvocine algae. As a result, the genomes of member species remarkably similar, suggesting the transition to multicellularity only requires the evolution of a few genes. The volvocine algae include members that span the range of morphological complexity from unicellular (e.g. Chlamydomonas reinhardtii) to undifferentiated multicellular (e.g. Gonium pectorale), to species with differentiated tissues (e.g. Volvox carteri). Corresponding to the morphological phenotypes, members of these organisms range from a simplified cell wall in the unicellular species into an expanded extracellular matrix important for multicellular group formation. To find genes important for multicellularity in undifferentiated multicellular Gonium pectorale, we performed a forward genetic screen for unicellular mutants. From this we identified two mutants, uc-1C7, where 99.6% of cells are unicellular and uc-1H7 where 95% of cells are unicellular. Both mutants were found to be sensitive to detergent lysis suggesting these mutants have defects in extracellular matrix assembly. Additionally, total cell wall extracts were prepared from uc-1C7 and wild-type strains. These were subjected to tandem mass spectrometry to identify which proteins were present in both lines, which demonstrated that the uc-1C7 mutant is missing major components of its cell wall. Using an antibody that is specific for Chlamydomonas cell walls, preliminary immunofluorescence tests show a reduced signal in the uc-1C7 and uc-1H7 mutants compared to wild-type. Our results point to two unicellular Gonium mutants that have defects in assembling a functional extracellular matrix. Because these mutants have a unicellular phenotype, it demonstrates that cell wall of Gonium is essential for undifferentiated colony formation.