Overview

Our lab aims to understand how regulatory networks are organized such that cells can process information, make decisions, and control their behavior. To address this problem, we study the genetic circuitry controlling cell cycle progression and cellular asymmetry in the bacterium Caulobacter crescentus. We use a combination of genetics, biochemistry, microscopy, genomics, and computational tools to map the regulatory network controlling the Caulobacter cell cycle and to explore, at a systems-level, the dynamics and design principles of this network. We are also beginning to examine mechansims by which cells maintain the specificity and fidelity of signal transduction systems in order to prevent unwanted cross-talk.

Research Summary

Cell cycle progression and the establishment of cellular asymmetry:
Caulobacter crescentus is a powerful model for studying questions of regulation as cells are easily synchronized, cell cycle progression can be tracked by monitoring a series of morphological transitions, and a complete suite of genetic tools is available. Although many of the major regulators in Caulobacter are known, it remains a major challenge to identify their connectivity and to understand the complete circuit which accounts for cell cycle oscillations.

Our major focus right now is understanding regulation by two-component signal transduction systems, one of the major classes of signaling molecules in bacteria. These systems are comprised of sensor histidine kinases and their response regulator substrates which execute changes in cellular physiology upon phosphorylation. The Caulobacter genome encodes 64 histidine kinases and 42 response regulators. At least 10 of these two-component genes are involved in cell cycle progression. This includes CtrA, the master regulator of the Caulobacter cell cycle, which is analogous to (although not homologous to) the eukaryotic cyclin-dependent kinases. CtrA is a transcription factor which directly regulates nearly 100 genes and which also binds to and represses the origin of replication. Hence, CtrA activity must be temporarily eliminated at the G1-S transition to permit DNA replication, but must rapidly reaccumulate afterwards to drive transcription in the late stages of the cell cycle.

We have recently mapped an integrated genetic circuit which can account for the changes in CtrA activity during cell cycle progression. This circuit incorporates all previous identified cell cycle regulators in Caulobacter and suggests a model for how oscillations are produced. Similar to other genetic oscillators, the circuit requires a delayed negative feedback loop. As CtrA accumulates it triggers its own destruction by inducing the down-regulation of its own upstream kinase, CckA, but is delayed in doing so until after cell division.

Crucial to the operation of this cell cycle circuit is the dynamic sub-cellular localization of several histidine kinases. This includes CckA which is normally located at one or both poles of the cell, but is temporarily dispersed throughout the membrane at the onset of S phase. We have identified several regulatory molecules which mediate this delocalization and are currently investigating the mechanism by which they control the localization of CckA.

We are also beginning to probe the dynamics and feedback structure of the cell cycle regulatory network. Why is the circuit so complex? What is the role of specific feedback loops to the reliability or robustness of the system? Is cell division the key time-delay necessary for oscillations? We are using a combination of genetics and biochemistry, as well as fluorescence microscopy of individual cells, to address these questions.

Stress, checkpoints, and genome stability:
We are also interested in understanding how cells sense and respond to changes in their environment. In particular, we focus on how Caulobacter cells respond to DNA damage. We have recently begun mapping the mechanisms by which cells sense DNA damage and respond by halting cell cycle progression and repairing their DNA. Our results thus far indicate that Caulobacter cells simultaneously up-regulate the genes required for physical repair of damaged DNA and down-regulate the master cell cycle regulator CtrA. The latter involves an unknown, post-translational mechanism which we are actively pursuing. In addition, we have recently identified the first bona fide checkpoint system in Caulobacter. This checkpoint is activated after DNA damage and helps halt the cell cycle by inhibiting chromosome segregation enzymes such as topo IV and gyrase. The precise mechanism by which this checkpoint acts is still being investigated.

Specificity in signal transduction systems:
Another major focus in the lab is understanding how cells maintain the specificity of signaling systems. Given the highly related nature of the two-component signaling proteins in bacteria, how do cells maintain the insulation of different pathways? What prevents harmful cross-talk? How are signals integrated? We use both computational and experimental approaches to answer these questions.

We have found that histidine kinases exhibit a strong, system-wide kinetic preference in vitro for their in vivo substrate response regulators. This suggests that specificity in two-component signaling systems is intrinsic to the molecules and that additional factors, such as scaffolds, could enhance specificity but are not essential. To map the domains and amino acids which dictate kinase specificity we are examining the behavior of chimeric kinases and using a variety of mutagenesis techniques. In addition we have looked for amino acids in cognate pairs of histidine kinases and response regulators which co-evolve. Using the results of these studies we are attempting to “rewire” signaling pathways, both as a test of how well we understand specificity and potentially for the design of bacteria with novel signaling capabilities. Identifying the molecular basis of kinase specificty will also enable us to investigate the evolution of signal transduction systems and the selective forces which shape large, paralogous gene families.

Selected Publications

Biondi, E. G., Reisinger, S. J., Skerker, J. M., Arif, M., Perchuk, B. S., Ryan, K. R., Laub, M. T. (2006) “Regulation of the Bacterial Cell Cycle by an Integrated Genetic Circuit” Nature, 444, 899-904.

Laub, M. T., Biondi, E. G., Skerker, J. M. (2006) “Systematic Mapping of Two-Component Signal Transduction Pathways and Phosphorelays” Methods in Enzymology, in press.

Biondi, E. G., Skerker, J. M., Arif, M., Prasol, M. S., Perchuk, B. S., Laub, M. T. (2006) “A Phosphorelay System Controls Stalk Biogenesis During Cell Cycle Progression in Caulobacter crescentus” Molecular Microbiology, 59, p. 386-401.

Skerker, J. M., Prasol, M., Perchuk, B., Biondi, E., Laub, M. T. (2005) “Two-Component Signal Transduction Pathways Regulating Growth and Cell Cycle Progression in a Bacterium: A System-Level Analysis” PLoS Biology, 3, p. 334-353.

Skerker, J. M., Laub, M. T. (2004) “Cell Cycle Progression and the Generation of Asymmetry in Caulobacter crescentus” Nature Reviews Microbiology, 2, p. 325-37.

Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. (2002) “Genes Directly Controlled by CtrA, a Master Regulator of the Caulobacter Cell Cycle”, Proc Natl Acad Sci USA, 99, p. 4632-37.

Laub, M. T., McAdams, H. H., Feldblyum, T., Fraser, C. M., Shapiro, L. (2000) “Global Analysis of the Genetic Network Controlling a Bacterial Cell Cycle” Science 290, p. 2144-2148.

Search PubMed for Laub lab publications.