Inappropriate Cts1 secretion causes premature septum degradation leading to failed abscission and thinned septa. (A) Inappropriate Cts1 secretion destroys chitin in the aberrant septum. The indicated genotypes, treated with or without auxin, were synchronized and released at 30°C. Cells were collected at times to enrich for cells completing cytokinesis (see Materials and methods). Calcofluor staining of fixed cells demonstrates chitin content. Enlarged region highlights bud neck chitin. Triangle highlights region of increased chitin; arrow, decreased chitin; asterisk, aberrant bud growth. Representative images are shown. Bar, 5 µm. (B) Inappropriate secretion of Cts1 leads to failed abscission and budding defects. Synchronized and auxin-treated inn1-AID and inn1-AID fir1Δ cells were released from arrest at 30°C for 3 h. Budding morphology was binned into chain-like or zygote-like, and the percentage of cells exhibiting the indicated morphology (representative image scale bar, 5 µm) is shown. Error bars represent SD of three independent experiments (n > 100 each trial); ***, P < 0.001 (two-tailed t test). (C) Strains from A were transformed to express the PH-domain of phospholipase C fused to GFP to mark the plasma membrane. Cells were collected as in A, and representative images of the indicated genotype with or without auxin are shown. The asterisk indicates a dead cell. Bar, 5 µm. (D) Cells inappropriately secreting Cts1 fail to complete septation or have thinned septal regions. Synchronized inn1-AID and inn1-AID fir1Δ cells were treated as in A and were processed for electron microscopy. Representative images of each genotype are shown. Bar, 1 µm.