Alexi I. Goranov

Measurement of mass, density, and volume during the cell cycle of yeast

Cell growth comprises changes in both mass and volume—two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell’s three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications.

The rate of cell growth is governed by cell cycle stage

Cell growth is an essential requirement for cell cycle progression. While it is often held that growth is independent of cell cycle position, this relationship has not been closely scrutinized. Here we show that in budding yeast, the ability of cells to grow changes during the cell cycle. We find that cell growth is faster in cells arrested in anaphase and G1 than in other cell cycle stages. We demonstrate that the establishment of a polarized actin cytoskeleton-either as a consequence of normal cell division or through activation of the mating pheromone response-potently attenuates protein synthesis and growth. We furthermore show by population and single-cell analysis that growth varies during an unperturbed cell cycle, slowing at the time of polarized growth. Our study uncovers a fundamental relationship whereby cell cycle position regulates growth.

Characterization of the Global Transcriptional Responses to Different Types of DNA Damage and Disruption of Replication in Bacillus subtilis

DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress

yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other Gram‐positive species. YabA interacts with the β‐processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We found that YabA function is mainly limited to replication initiation at oriC. Loss of YabA did not significantly alter expression of genes controlled by DnaA during exponential growth or after replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity. We also found that DnaN activates replication initiation apparently through effects on YabA. Furthermore, association of GFP‐YabA with the replisome correlated with the presence of DnaN at replication forks, but was independent of DnaA. Our results are consistent with models in which YabA inhibits replication initiation at oriC, and perhaps DnaA function at oriC, but not with models in which YabA generally modulates the activity of DnaA in response to replication stress.

Growth and division--not a one-way road.

Maintaining cell size homeostasis and regulating cell size in response to changing conditions is a fundamental property of organisms. Here we examine the recent advances in our understanding of the interplay between accumulation of mass (growth) and the progression through the cell cycle (proliferation), the coordination of which determines the size of cells. It is well established that growth affects cell division (reviewed in Jorgensen and Tyers, 2004). This review will focus on the reverse, less well-defined relationship-how cell cycle progression affects growth. We will summarize findings that indicate that growth is not constant during the cell cycle and discuss the surprising possibility that cyclin-dependent kinases (CDKs) inhibit growth.

Changes in cell morphology are coordinated with cell growth through the TORC1 pathway.

BACKGROUND Growth rate is determined not only by extracellular cues such as nutrient availability but also by intracellular processes. Changes in cell morphology in budding yeast, mediated by polarization of the actin cytoskeleton, have been shown to reduce cell growth. RESULTS Here we demonstrate that polarization of the actin cytoskeleton inhibits the highly conserved Target of Rapamycin Complex 1 (TORC1) pathway. This downregulation is suppressed by inactivation of the TORC1 pathway regulatory Iml1 complex, which also regulates TORC1 during nitrogen starvation. We further demonstrate that attenuation of growth is important for cell recovery after conditions of prolonged polarized growth. CONCLUSIONS Our results indicate that extended periods of polarized growth inhibit protein synthesis, mass accumulation, and the increase in cell size at least in part through inhibiting the TORC1 pathway. We speculate that this mechanism serves to coordinate the ability of cells to increase in size with their biosynthetic capacity.