Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance

Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism. We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle. Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations, suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways. We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments. These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors, leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission. Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures. DOI: http://dx.doi.org/10.7554/eLife.02678.00

The dynamics of enzymatic switch cascades

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Physics; and, (S.B.)--Massachusetts Institute of Technology, Dept. of Mathematics, 2004.Includes bibliographical references (leaf 67).We examine the dynamics of the mitogen-activated protein kinase (MAPK) multi-step enzymatic switching cascade, a highly conserved architecture utilised in cellular signal transduction. In treating the equations of motion, we replace the usual deterministic differential equation formalism with stochastic equations to accurately model the 'effective collisions' picture of the biochemical reactions that constitute the network. Furthermore we measure the fidelity of the signaling process through the mutual information content between the output of a given switch and the original environmental input to the system. We find that the enzymatic switches act as low-pass filters, with each switch in the cascade able to average over high frequency stochastic fluctuations in the network and throughput cleaner signals to downstream switches. We find optimal regions of mutual information transfer with respect to reaction velocity and species number parameters, and observe the dynamical memory-gain and memory-loss as well as decay in mutual information in quadruple-linked switch systems.by Shankar Mukherji.S.B

Robust Circadian Oscillations in Growing Cyanobacteria Require Transcriptional Feedback

The remarkably stable circadian oscillations of single cyanobacteria enable a population of growing cells to maintain synchrony for weeks. The cyanobacterial pacemaker is a posttranslational regulation (PTR) circuit that generates circadian oscillations in the phosphorylation state of the clock protein KaiC. Layered on top of the PTR is transcriptional-translational feedback regulation (TTR), common to all circadian systems, consisting of a negative feedback loop in which KaiC regulates its own production. We found that the PTR circuit is sufficient to generate oscillations in growing cyanobacteria. However, in the absence of TTR, individual oscillators were less stable and synchrony was not maintained in a population of cells. Experimentally constrained mathematical modeling reproduced sustained oscillations in the PTR circuit alone and demonstrated the importance of TTR for oscillator synchrony.Chemistry and Chemical BiologyMolecular and Cellular BiologyPhysic

MicroRNAs can generate thresholds in target gene expression

MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.National Institutes of Health (U.S.). Director's Pioneer Award (1DP1OD003936)National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874)United States. Public Health Service (Grant R01-CA133404)United States. Public Health Service (Grant R01-GM34277)National Cancer Institute (U.S.) (PO1-CA42063)National Cancer Institute (U.S.) Cancer Center Support (Grant P30-CA14051)Howard Hughes Medical Institute. Predoctoral FellowshipCleo and Paul Schimmel Foundation. FellowshipNatural Sciences and Engineering Research Council of Canada PGS Scholarshi

Synthetic biology: Understanding biological design from synthetic circuits

An important aim of synthetic biology is to uncover the design principles of natural biological systems through the rational design of gene and protein circuits. Here, we highlight how the process of engineering biological systems — from synthetic promoters to the control of cell–cell interactions — has contributed to our understanding of how endogenous systems are put together and function. Synthetic biological devices allow us to grasp intuitively the ranges of behaviour generated by simple biological circuits, such as linear cascades and interlocking feedback loops, as well as to exert control over natural processes, such as gene expression and population dynamics

Robustness and tunability in biological systems

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 123-139).Cells face a core tension between studiously preventing change in certain properties from extrinsic perturbations while allowing other properties to be tuned. One way cells have resolved this tension is to utilize systems that are both robust and tunable. Systems can achieve this through network design, which can contain submodules that are themselves either robust or tunable, or through network components that are robust over only a defined set of parameter ranges. This work examines these two categories with two specific examples described below. To explore how a simple network can be both robust and tunable, we make use of the osmosensing pathway in the budding yeast Saccharomyces cerevisiae. The pathway consists of two modules: a phosphorelay module that senses the osmotic shock signal that feeds into a mitogen-activated protein kinase (MAPK) module. Using a combination of systematic complementation experiments and computational sensitivity analysis, we show that the phosphorelay module is robust to changes in the kinetic parameters characterizing signal propagation through the module while signaling through the MAPK module can be tuned by changing the rate constants. Furthermore, we show that pathway robustness to rate constant changes has consequences for the evolvability of the osmosensing cascade. Populations of yeast cells challenged to alter the input/output relationship of the cascade saw their MAPK proteins preferentially targeted by natural selection over their phosphorelay counterparts. To explore how a simple regulatory element can be both robust and tunable, we turn our attention to gene regulation by microRNA (miRNA). MiRNAs are short regulatory RNA molecules that repress gene expression in a sequence-dependent manner. By observing the strength of miRNA-mediated repression in individual cells, we show that the strength of repression depends strongly on the relative abundance of the miRNA and its target. Below a threshold level of target message miRNA robustly silences the conversion of mRNA input into protein output, but above this threshold miRNAmediated repression generates an ultrasensitive response to mRNA input allowing the strength of repression to be tuned over a wide variety of values.Ph.D