Our lab aims to understand how cells process information, make decisions, and control their own behavior. This information-processing capability is ultimately endowed by complex regulatory networks and signal transduction pathways, but the design and operating principles of such molecular-based circuits remain poorly understood. We are tackling this challenge in two particular contexts. First, we are dissecting the genetic circuitry that controls cell cycle progression and the generation of cellular asymmetry in the bacterium Caulobacter crescentus. For this work, we combine genetics, biochemistry, microscopy, genomics, and computational tools to map the regulatory network controlling the cell cycle and to explore how the molecules involved are connected and orchestrated at the systems-level. Second, we are studying the mechanisms by which cells maintain the specificity and fidelity of signal transduction systems in order to prevent unwanted cross-talk. These studies include efforts in protein engineering and the design of synthetic signaling circuits.
Scanning electron micrographs of wild type Caulobacter crescentus showing the generation of two different daughter cells following an asymmetric cell division.
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 that directly regulates nearly 100 genes and that also binds to and represses the origin of replication. Hence, CtrA activity must cycle between periods of inactivity that permit DNA replication and periods of activity during which it drives transcription of key cell cycle genes.
We have recently mapped, for the first time, an integrated genetic circuit that 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 eventually 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 control of CckA activity via an important cofactor. We are currently characterizing how this cofactor mediates changes in CckA activity and sub-cellular localization to mediate switch-like transitions in the cell cycle.
We are also probing the 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? We are using a combination of genetics and biochemistry, as well as fluorescence microscopy of individual cells, to address these questions. In addition, our recent data suggests the existence of two interlinked, but separate oscillators that collaborate to drive the cell cycle. We are actively investigating the nature of each oscillator and the mechanisms that synchronize them.
Stress, checkpoints, and genome stability
We are also interested in understanding how cells sense and respond to changes in their environment. We have recently focused on the mechanisms by which cells sense DNA damage and respond by halting cell cycle progression and repairing their DNA. Our results have led to the identification of the first bona fide cell cycle checkpoint in Caulobacter. We are characterizing the molecular basis of this surveillance system and the means by which it inhibits cell division following DNA damage. We have also uncovered evidence of a new pathway that regulates the activity of CtrA, the master cell cycle regulator.
Specificity in signal transduction systems
Another major focus in the lab is to understand 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. Our goal is to develop a detailed, atomic-level understanding of specificity that is sufficient to guide the rational rewiring of two-component signaling pathways.
We have found that histidine kinases exhibit a strong, system-wide kinetic preference in vitro for their in vivo substrate response regulators. This finding indicates that the specificity of two-component signaling systems is intrinsic to the molecules involved and that any additional factors, such as scaffolds, could enhance specificity but are not essential. To map the domains and amino acids which dictate kinase specificity we have analyzed patterns of amino-acid coevolution in large sets of cognate kinase-substrate pairs. These coevolution analyses are coupled with mutagenesis and biochemical studies to understand how specificity is encoded at the amino-acid level. Guided by these studies we are rationally “rewiring” signaling pathways, both as a stringent test of how well we understand specificity and for constructing synthetic signaling pathways in vivo. Finally, our work on kinase specificity is enabling us to probe molecular evolution and the selective forces that shape large, paralogous families of signal transduction systems.
Skerker, J.M., Perchuk, B.S., Siryaporn, A., Lubin, E., Ashenberg, O., Goulian, M., Laub, M.T. (2008) “Rewiring the specificity of two-component signal transduction systems”, Cell, 133, p. 1043-1054.
Laub, M.T., Goulian, M. (2007) “Specificity in two-component signal transduction systems”, Annual Review of Genetics, 41, p. 121-145.
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, p. 899-904.
Biondi, E.G., Skerker, J.M., Perchuk, B., Prasol, M., 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.