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In JoVE (1)
Other Publications (12)
- Proceedings of the National Academy of Sciences of the United States of America
- Brain, Behavior and Evolution
- The Journal of Cell Biology
- Biophysical Journal
- Journal of Molecular Biology
- Current Biology : CB
- The Journal of Cell Biology
- Proceedings of the National Academy of Sciences of the United States of America
- Science (New York, N.Y.)
- Biophysical Journal
- Journal of Cell Science
- Wiley Interdisciplinary Reviews. Systems Biology and Medicine
Articles by Hao Yuan Kueh in JoVE
JoVE General
Monitoring Actin Disassembly with Time-lapse Microscopy
Dept. of Systems Biology, Harvard Medical School
Other articles by Hao Yuan Kueh on PubMed
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.
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.
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.
Intrinsic Fluctuations, Robustness, and Tunability in Signaling Cycles
Covalent modification cycles (e.g., phosphorylation-dephosphorylation) underlie most cellular signaling and control processes. Low molecular copy number, arising from compartmental segregation and slow diffusion between compartments, potentially renders these cycles vulnerable to intrinsic chemical fluctuations. How can a cell operate reliably in the presence of this inherent stochasticity? How do changes in extrinsic parameters lead to variability of response? Can cells exploit these parameters to tune cycles to different ranges of stimuli? We study the dynamics of an isolated phosphorylation cycle. Our model shows that the cycle transmits information reliably if it is tuned to an optimal parameter range, despite intrinsic fluctuations and even for small input signal amplitudes. At the same time, the cycle is sensitive to changes in the concentration and activity of kinases and phosphatases. This sensitivity can lead to significant cell-to-cell response variability. It also provides a mechanism to tune the cycle to transmit signals in various amplitude ranges. Our results show that signaling cycles possess a surprising combination of robustness and tunability. This combination makes them ubiquitous in eukaryotic signaling, optimizing signaling in the presence of fluctuations using their inherent flexibility. On the other hand, cycles tuned to suppress intrinsic fluctuations can be vulnerable to changes in the number and activity of kinases and phosphatases. Such trade-offs in robustness to intrinsic and extrinsic fluctuations can influence the evolution of signaling cascades, making them the weakest links in cellular circuits.
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.
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.
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 approximately 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.
Structural Plasticity in Actin and Tubulin Polymer Dynamics
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.
Quantitative Analysis of Actin Turnover in Listeria Comet Tails: Evidence for Catastrophic Filament Turnover
Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.
Actin Behavior in Bulk Cytoplasm is Cell Cycle Regulated in Early Vertebrate Embryos
The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP-Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.
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.
