Hao Yuan Kueh

Mechanisms of noise-resistance in genetic oscillators

A wide range of organisms use circadian clocks to keep internal sense of daily time and regulate their behavior accordingly. Most of these clocks use intracellular genetic networks based on positive and negative regulatory elements. The integration of these “circuits” at the cellular level imposes strong constraints on their functioning and design. Here, we study a recently proposed model [Barkai, N. & Leibler, S. (2000) Nature (London), 403, 267–268] that incorporates just the essential elements found experimentally. We show that this type of oscillator is driven mainly by two elements: the concentration of a repressor protein and the dynamics of an activator protein forming an inactive complex with the repressor. Thus, the clock does not need to rely on mRNA dynamics to oscillate, which makes it especially resistant to fluctuations. Oscillations can be present even when the time average of the number of mRNA molecules goes below one. Under some conditions, this oscillator is not only resistant to but, paradoxically, also enhanced by the intrinsic biochemical noise.

Structural Plasticity in Actin and Tubulin Polymer Dynamics

Discussing Polymerization Dynamics Actin and tubulin represent the major components of microfilaments and microtubules, which are responsible for the cytoskeletal framework of cells. Together with their recently discovered bacterial counterparts, the mechanisms of assembly and disassembly of polymers of these proteins are fundamental to our understanding of cytoplasmic organization and motility. Kueh and Mitchison (p. 960) review mechanisms of cytoskeleton polymerization dynamics and discuss the relative importance of structural plasticity in regulating polymerization. Actin filaments and microtubules polymerize and depolymerize by adding and removing subunits at polymer ends, and these dynamics drive cytoplasmic organization, cell division, and cell motility. Since Wegner proposed the treadmilling theory for actin in 1976, it has largely been assumed that the chemical state of the bound nucleotide determines the rates of subunit addition and removal. This chemical kinetics view is difficult to reconcile with observations revealing multiple structural states of the polymer that influence polymerization dynamics but that are not strictly coupled to the bound nucleotide state. We refer to these phenomena as “structural plasticity” and discuss emerging evidence that they play a central role in polymer dynamics and function.

Positive Feedback Between PU.1 and the Cell Cycle Controls Myeloid Differentiation

A Different Cycle for Differentiation The regulated expression of transcription factors determines cell fate decisions during cell differentiation. The transcription factor PU.1 is an important determinant in the differentiation of hematopoietic progenitors to lymphocytes or myeloid cells, where high expression induces macrophage differentiation, whereas low expression leads to the development of B lymphocytes. How PU.1 expression levels are regulated during this cell fate choice, however, is not well understood. Kueh et al. (p. 670, published online 18 July) found that in mice, reduced transcription of PU.1 led to its reduced expression in developing B lymphocytes, whereas in macrophages, PU.1 was able to accumulate stably because of a lengthening of the cell cycle. Exogenous expression of PU.1 in progenitors supported cell cycle lengthening and macrophage differentiation, and mathematical modeling suggested that such a feedback loop could maintain a slow-dividing macrophage developmental state. Regulation of cell cycle length is a feedback mechanism that controls cell fate decisions in developing macrophages. Regulatory gene circuits with positive-feedback loops control stem cell differentiation, but several mechanisms can contribute to positive feedback. Here, we dissect feedback mechanisms through which the transcription factor PU.1 controls lymphoid and myeloid differentiation. Quantitative live-cell imaging revealed that developing B cells decrease PU.1 levels by reducing PU.1 transcription, whereas developing macrophages increase PU.1 levels by lengthening their cell cycles, which causes stable PU.1 accumulation. Exogenous PU.1 expression in progenitors increases endogenous PU.1 levels by inducing cell cycle lengthening, implying positive feedback between a regulatory factor and the cell cycle. Mathematical modeling showed that this cell cycle–coupled feedback architecture effectively stabilizes a slow-dividing differentiated state. These results show that cell cycle duration functions as an integral part of a positive autoregulatory circuit to control cell fate.

Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

Rapid actin monomer–insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.

Brain Architecture and Social Complexity in Modern and Ancient Birds

Vertebrate brains vary tremendously in size, but differences in form are more subtle. To bring out functional contrasts that are independent of absolute size, we have normalized brain component sizes to whole brain volume. The set of such volume fractions is the cerebrotype of a species. Using this approach in mammals we previously identified specific associations between cerebrotype and behavioral specializations. Among primates, cerebrotypes are linked principally to enlargement of the cerebral cortex and are associated with increases in the complexity of social structure. Here we extend this analysis to include a second major vertebrate group, the birds. In birds the telencephalic volume fraction is strongly correlated with social complexity. This correlation accounts for almost half of the observed variation in telencephalic size, more than any other behavioral specialization examined, including the ability to learn song. A prominent exception to this pattern is owls, which are not social but still have very large forebrains. Interpolating the overall correlation for Archaeopteryx, an ancient bird, suggests that its social complexity was likely to have been on a par with modern domesticated chickens. Telencephalic volume fraction outperforms residuals-based measures of brain size at separating birds by social structure. Telencephalic volume fraction may be an anatomical substrate for social complexity, and perhaps cognitive ability, that can be generalized across a range of vertebrate brains, including dinosaurs.

Dynamic stabilization of actin filaments.

We report here that actin filaments in vitro exist in two populations with significantly different shrinkage rates. Newly polymerized filaments shrink rapidly, primarily from barbed ends, at 1.8/s, but as they age they switch to a stable state that shrinks slowly, primarily from pointed ends, at ≈0.1/s. This dynamic filament stabilization runs opposite to the classical prediction that actin filaments become more unstable with age as they hydrolyze their bound ATP and release phosphate. Upon cofilin treatment, aged filaments revert to a dynamic state that shows accelerated shrinkage from both ends at a combined rate of 5.9/s. In light of recent electron microscopy studies [Orlova A, et al. (2004) Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization. Proc Natl Acad Sci USA 101:17664–17668], we propose that dynamic stabilization arises from rearrangement of the filament structure from a relatively disordered state immediately after polymerization to the canonical Holmes helix, a change that is reversed by cofilin binding. Our results suggest that plasticity in the internal structure of the actin filament may play a fundamental role in regulating actin dynamics and may help cells build actin assemblies with vastly different turnover rates.

Coronin-1A stabilizes F-actin by bridging adjacent actin protomers and stapling opposite strands of the actin filament.

Coronins are F-actin-binding proteins that are involved, in concert with Arp2/3, Aip1, and ADF/cofilin, in rearrangements of the actin cytoskeleton. An understanding of coronin function has been hampered by the absence of any structural data on its interaction with actin. Using electron microscopy and three-dimensional reconstruction, we show that coronin-1A binds to three protomers in F-actin simultaneously: it bridges subdomain 1 and subdomain 2 of two adjacent actin subunits along the same long-pitch strand, and it staples subdomain 1 and subdomain 4 of two actin protomers on different strands. Such a mode of binding explains how coronin can stabilize actin filaments in vitro. In addition, we show which residues of F-actin may participate in the interaction with coronin-1A. Human nebulin and Xin, as well as Salmonella invasion protein A, use a similar mechanism to stabilize actin filaments. We suggest that the stapling of subdomain 1 and subdomain 4 of two actin protomers on different strands is a common mechanism for F-actin stabilization utilized by many actin-binding proteins that have no homology.

Spatial patterning of metabolism by mitochondria, oxygen, and energy sinks in a model cytoplasm.

Metabolite gradients might guide mitochondrial localization in cells and angiogenesis in tissues. It is unclear whether they can exist in single cells, because the length scale of most cells is small compared to the expected diffusion times of metabolites. For investigation of metabolic gradients, we need experimental systems in which spatial patterns of metabolism can be systematically measured and manipulated. We used concentrated cytoplasmic extracts from Xenopus eggs as a model cytoplasm, and visualized metabolic gradients formed in response to spatial stimuli. Restriction of oxygen supply to the edge of a drop mimicked distance to the surface of a single cell, or distance from a blood vessel in tissue. We imaged a step-like increase of Nicotinamide adenine dinucleotide (NAD) reduction approximately 600 microm distant from the oxygen source. This oxic-anoxic switch was preceded on the oxic side by a gradual rise of mitochondrial transmembrane potential (Deltapsi) and reactive oxygen species (ROS) production, extending over approximately 600 microm and approximately 300 microm, respectively. Addition of Adenosine triphosphate (ATP)-consuming beads mimicked local energy sinks in the cell. We imaged Deltapsi gradients with a decay length of approximately 50-300 microm around these beads, in the first visualization of an energy demand signaling gradient. Our study demonstrates that mitochondria can pattern the cytoplasm over length scales that are suited to convey morphogenetic information in large cells and tissues and provides a versatile model system for probing of the formation and function of metabolic gradients.

Regulatory gene network circuits underlying T cell development from multipotent progenitors

Regulatory gene circuits enable stem and progenitor cells to detect and process developmental signals and make irreversible fate commitment decisions. To gain insight into the gene circuits underlying T cell fate decision making in progenitor cells, we generated an updated T‐lymphocyte developmental gene regulatory network from genes and connections found in the literature. This reconstruction allowed us to identify candidate regulatory gene circuit elements underlying T cell fate decision making. Here, we examine the roles of these circuits in facilitating different aspects of the decision making process, and discuss experiments to further probe their structure and function. WIREs Syst Biol Med 2012, 4:79–102. doi: 10.1002/wsbm.162