Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway

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Abstract
Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription. All cell types have a characteristic size, but the means by which cell size is determined remain mysterious. In proliferating cells, control mechanisms termed checkpoints are thought to prevent cells from dividing until they have reached a minimum size, but the nature of size checkpoints has proved difficult to dissect. The unicellular alga Chlamydomonas reinhardtii divides via an unusual mechanism that uncouples growth from division, and thereby allows a direct assessment of how different genetic pathways contribute to size control. The retinoblastoma (RB) tumor suppressor pathway is a critical regulator of cell cycle control in plants and animals and is thought to act as a transcriptional switch for cell cycle genes, but it had not been directly implicated in cell size checkpoint function. The authors found that mutations in genes that encode key proteins of the RB pathway in Chlamydomonas affect cell size and cell cycle control by altering size checkpoint function. Unexpectedly, the predicted transcriptional targets of the RB pathway were not affected by the mutations, and blocking transcription did not alter cell size control. These data link the RB tumor suppressor pathway directly to size control and suggest the possibility that cell size and cell cycle control by the RB pathway may not be coupled to its transcriptional output.