Lucy Shapiro, Ph.D.

Professor of Developmental Biology

Our goal is to define the complete genetic circuitry that coordinates cells differentiation as a function of the cell cycle. We have recently shown that both chromosomal regions and regulatory proteins exhibit dynamic localization during the cell cycle. Thus, deciphering the entire regulatory network is akin to playing 3-dimensional chess.

Our model cell is the differentiating bacterium, Caulobacter. This microbe has a fully annotated genome of only 3767 genes with a clearly defined cell cycle. Full genome microarray analysis has shown that the transcription of 575 genes (19% of the genome) is cell cycle controlled. We found that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding protein that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes. Genetic and biochemical analysis of the cell cycle regulatory factors revealed that both cyclic phosphorylation cascades and proteolysis are critical determinants of bacterial cell cycle control in a manner analogous to the control of the eukaryotic cell cycle. The prokaryotic and eukaryotic proteins differ, but the paradigm has been conserved.

To understand chromosome dynamics and DNA replication as a function of the cell cycle, we have used fluorescence microscopy of living cells and FISH to examine chromosome and replisome movement. We have found that origin of chromosome replication resides at the cell pole and that the replisome assembles at that pole only during the G1-S transition when the cell becomes competent for the initiation of replication. The newly replicated origin rapidly moves to the opposite pole, while the replisome complex, as an untethered replication factory, is pushed towards the division plane. At the conclusion of replication, the replisome disassembles. The replisome then reforms at the origin only in the replication-competent stalked cell progeny and not in the swarmer progeny cell. We propose that newly replicated DNA is bidirectionally extruded form the replisome, contributing to chromosome segregation. Also contributing to chromosome condensation and segregation is the SMC [Structural Maintenance of Chromosomes] protein. An SMC null results in a cell cycle arrest just prior to cell division, suggesting a checkpoint that detects the completion of chromosome segregation before allowing cell division.