Fig. 1. Loss of FrmA impairs both cell shape and cell-substrate adhesion. (A) Single-cell-derived frmA^– clones were identified by PCR of genomic DNA. PCR of wild-type genomic DNA yielded a 2 kbp product (^*), whereas PCR of frmA^– genomic DNA yielded a 2.4 kbp product (^**). A primer set that amplified a 1 kbp fragment of eF1 was also included in the PCR reaction for FrmA as a control to show the specificity of FrmA disruption (+). At least three single-cell-derived frmA^– clones were isolated and the analysis of a representative frmA^– clone is shown. (B) Real-time RT-PCR was used to determine the expression of FrmA, talinB (example of a protein that is upregulated upon starvation) and eF1 (example of a protein that is not regulated by starvation) at 0 and 6 hours of starvation. Total RNA was isolated on two separate occasions and real-time RT-PCR reactions were carried out in triplicate. The average ± s.d. is shown. (C) Phase images of non-starved cells (40x objective used for all). Broad membrane protrusions (white arrows) and filopodia (yellow arrows) are indicated. (D) Cell adhesion to substrate was determined under conditions of increased shear stress. Experiments were carried out on at least four separate occasions in triplicate and the average ± s.e.m. is shown. (E) Adhesion of wild-type and frmA^– cells expressing GFP, GFP:FERM(1) or GFP:FERM(2).