Switch-like Manner Research Articles

Substrate Rigidity Modulates Segmentation Clock Dynamics in Isolated Presomitic Mesoderm Cells.

The segmentation clock, a genetic oscillator in the presomitic mesoderm (PSM), is known to be influenced by biochemical signals, yet its potential regulation by mechanical cues remains unclear. The complex PSM microenvironment has made it challenging to isolate the effects of mechanical perturbations on clock behavior. Here we investigated how mechanical stimuli affect clock oscillations by culturing zebrafish PSM cells on PDMS micropost arrays with tunable rigidities (0.6-1200 kPa). We observed an inverse sigmoidal relationship between surface rigidity and both the percentage of oscillating cells and the number of oscillation cycles, with a switching threshold between 3-6 kPa. The periods of oscillating cells showed a consistently broad distribution across rigidity changes. Moreover, these cells exhibited distinct biophysical properties, such as reduced motility, contractility, and sustained circularity. These findings highlight the crucial role of cell-substrate interactions in regulating segmentation clock behavior, providing insights into the mechanobiology of somitogenesis.

Tumor necrosis factor is a necroptosis-associated alarmin.

Necroptosis is a form of regulated cell death that can occur downstream of several immune pathways. While previous studies have shown that dysregulated necroptosis can lead to strong inflammatory responses, little is known about the identity of the endogenous molecules that trigger these responses. Using a reductionist in vitro model, we found that soluble TNF is strongly released in the context of necroptosis. On the one hand, necroptosis promotes TNF translation by inhibiting negative regulatory mechanisms acting at the post-transcriptional level. On the other hand, necroptosis markedly enhances TNF release by activating ADAM proteases. In studying TNF release at single-cell resolution, we found that TNF release triggered by necroptosis is activated in a switch-like manner that exceeds steady-state TNF processing in magnitude and speed. Although this shedding response precedes massive membrane damage, it is closely associated with lytic cell death. Further, we found that lytic cell death induction using a pore-forming toxin also triggers TNF shedding, indicating that the activation of ADAM proteases is not strictly related to the necroptotic pathway but likely associated with biophysical changes of the cell membrane upon lytic cell death. These results demonstrate that lytic cell death, particularly necroptosis, is a critical trigger for TNF release and thus qualify TNF as a necroptosis-associated alarmin.

Cofilin regulates actin network homeostasis and microvilli length in mouse oocytes.

How multiple actin networks coexist in a common cytoplasm while competing for a shared pool of monomers is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here, we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner upon meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread elongation of microvilli, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.

Bifurcation Analysis of a Heat-Sensitive Epigenetic Regulatory Network.

An epigenetic regulatory network that influences transgenerational inheritance of a heat-altered phenotype was recently discovered in Arabidopsis. Our analysis shows that transgenerational inheritance of the heat-altered phenotype operates in a switch-like manner and can be turned on or off as a function of heat. We also show that trans-acting small interfering RNAs act as an "inverse amplifier" of HTT5, the protein that controls the heat-altered phenotype by a currently unknown mechanism. Our analysis uses the resultant to find an analytic expression for a cusp curve in parameter space and to find a parameter bound on switch-like behavior.

TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor–associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans.

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential “prion-like” domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59’s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

Cellular memory in eukaryotic chemotaxis depends on the background chemoattractant concentration.

Cells of the social amoeba Dictyostelium discoideum migrate to a source of periodic traveling waves of chemoattractant as part of a self-organized aggregation process. An important part of this process is cellular memory, which enables cells to respond to the front of the wave and ignore the downward gradient in the back of the wave. During this aggregation, the background concentration of the chemoattractant gradually rises. In our microfluidic experiments, we exogenously applied periodic waves of chemoattractant with various background levels. We find that increasing background does not make detection of the wave more difficult, as would be naively expected. Instead, we see that the chemotactic efficiency significantly increases for intermediate values of the background concentration but decreases to almost zero for large values in a switch-like manner. These results are consistent with a computational model that contains a bistable memory module, along with a nonadaptive component. Within this model, an intermediate background level helps preserve directed migration by keeping the memory activated, but when the background level is higher, the directional stimulus from the wave is no longer sufficient to activate the bistable memory, suppressing directed migration. These results suggest that raising levels of chemoattractant background may facilitate the self-organized aggregation in Dictyostelium colonies.

Stochastic activation and bistability in a Rab GTPase regulatory network

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.

Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner.

The spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. Each mammalian kinetochore binds many microtubules, but how many attached microtubules are required to turn off the checkpoint, and how the kinetochore monitors microtubule numbers, are not known and are central to understanding SAC mechanisms and function. To address these questions, here we systematically tune and fix the fraction of Hec1 molecules capable of microtubule binding. We show that Hec1 molecules independently bind microtubules within single kinetochores, but that the kinetochore does not independently process attachment information from different molecules. Few attached microtubules (20% occupancy) can trigger complete Mad1 loss, and Mad1 loss is slower in this case. Finally, we show using laser ablation that individual kinetochores detect changes in microtubule binding, not in spindle forces that accompany attachment. Thus, the mammalian kinetochore responds specifically to the binding of each microtubule and counts microtubules as a single unit in a sensitive and switch-like manner. This may allow kinetochores to rapidly react to early attachments and maintain a robust SAC response despite dynamic microtubule numbers.

Spatiotemporally Controlled Multiplexed Photothermal Microfluidic Pumping under Monitoring of On-Chip Thermal Imaging.

Intelligent contactless microfluidic pumping strategies have been increasingly desirable for operation of lab-on-a-chip devices. Herein, we present a photothermal microfluidic pumping strategy for on-chip multiplexed cargo transport in a contactless and spatiotemporally controllable fashion based on the application of near-infrared laser-driven photothermal effect in microfluidic paper-based devices (μPDs). Graphene oxide (GO)-doped thermoresponsive poly(N-isopropylacrylamide)-acrylamide hydrogels served as the photothermally responsive cargo reservoirs on the μPDs. In response to remote contactless irradiation by an 808 nm laser, on-chip phase transition of the composite hydrogels was actuated in a switchlike manner as a result of the photothermal effect of GO, enabling robust on-chip pumping of cargoes from the hydrogels to predefined arrays of reaction zones. The thermal imaging technique was employed to monitor the on-chip photothermal pumping process. The microfluidic pumping performance can be spatiotemporally controlled in a quantitative way by remotely tuning the laser power, irradiation time, and GO concentration. The pumping strategy was exemplified by FeCl3 and horseradish peroxidase as the model cargoes to implement on-chip Prussian blue- and 3,3',5,5'-tetramethylbenzidine-based colorimetric reactions, respectively. Furthermore, multiplexed on-demand microfluidic pumping was achieved by flexibly adjusting the irradiation pathway and the microfluidic pattern. The new microfluidic pumping strategy shows great promise for diverse microfluidic applications due to its flexibility, high integratability into lab-on-a-chip devices, and contactless and spatiotemporal controllability.

Excitation Modulation of Upconversion Nanoparticles for Switch-like Control of Ultraviolet Luminescence.

The ability to control ultraviolet (UV) luminescence intensity in a switch-like manner is demonstrated through the use of 980 nm excitation pulse-width modulation in NaYF4:Yb3+,Tm3+ upconversion nanoparticles (UNPs). Varying the ytterbium doping resulted in a single order of magnitude improvement of UV luminescence intensity. The excitation pulse-width modulation technique applied to these optimized UNPs enables 3 orders of magnitude control over UV luminescence intensity while maintaining NIR luminescence emission at 800 nm. Controlled in the switch-like manner, these UNPs can transfer their UV energy to 9,10-diphenylanthracene (DPA). Independent control of NIR luminescence and UV energy transfer through NIR excitation modulation may find applications in the development of multifunctional theranostic systems.

Distinct NF-κB and MAPK Activation Thresholds Uncouple Steady-State Microbe Sensing from Anti-pathogen Inflammatory Responses

The innate immune system distinguishes low-level homeostatic microbial stimuli from those of invasive pathogens, yet we lack understanding of how qualitatively similar microbial products yield context-specific macrophage functional responses. Using quantitative approaches, we found that NF-κB and MAPK signaling was activated at different concentrations of a stimulatory TLR4 ligand in both mouse and human macrophages. Above a threshold of ligand, MAPK were activated in a switch-like manner, facilitating production of inflammatory mediators. At ligand concentrations below this threshold, NF-κB signaling occurred, promoting expression of a restricted set of genes and macrophage priming. Among TLR-induced genes, we observed an inverse correlation between MAPK dependence and ligand sensitivity, highlighting the role of this signaling dichotomy in partitioning innate responses downstream of a single receptor. Our study reveals an evolutionarily conserved innate immune response system in which danger discrimination is enforced by distinct thresholds for NF-κB and MAPK activation, which provide sequential barriers to inflammatory mediator production.

Dynamical Allocation of Cellular Resources as an Optimal Control Problem: Novel Insights into Microbial Growth Strategies.

Microbial physiology exhibits growth laws that relate the macromolecular composition of the cell to the growth rate. Recent work has shown that these empirical regularities can be derived from coarse-grained models of resource allocation. While these studies focus on steady-state growth, such conditions are rarely found in natural habitats, where microorganisms are continually challenged by environmental fluctuations. The aim of this paper is to extend the study of microbial growth strategies to dynamical environments, using a self-replicator model. We formulate dynamical growth maximization as an optimal control problem that can be solved using Pontryagin’s Maximum Principle. We compare this theoretical gold standard with different possible implementations of growth control in bacterial cells. We find that simple control strategies enabling growth-rate maximization at steady state are suboptimal for transitions from one growth regime to another, for example when shifting bacterial cells to a medium supporting a higher growth rate. A near-optimal control strategy in dynamical conditions is shown to require information on several, rather than a single physiological variable. Interestingly, this strategy has structural analogies with the regulation of ribosomal protein synthesis by ppGpp in the enterobacterium Escherichia coli. It involves sensing a mismatch between precursor and ribosome concentrations, as well as the adjustment of ribosome synthesis in a switch-like manner. Our results show how the capability of regulatory systems to integrate information about several physiological variables is critical for optimizing growth in a changing environment.

Is the Cell Nucleus a Necessary Component in Precise Temporal Patterning?

One of the functions of the cell nucleus is to help regulate gene expression by controlling molecular traffic across the nuclear envelope. Here we investigate, via stochastic simulation, what effects, if any, does segregation of a system into the nuclear and cytoplasmic compartments have on the stochastic properties of a motif with a negative feedback. One of the effects of the nuclear barrier is to delay the nuclear protein concentration, allowing it to behave in a switch-like manner. We found that this delay, defined as the time for the nuclear protein concentration to reach a certain threshold, has an extremely narrow distribution. To show this, we considered two models. In the first one, the proteins could diffuse freely from cytoplasm to nucleus (simple model); and in the second one, the proteins required assistance from a special class of proteins called importins. For each model, we generated fifty parameter sets, chosen such that the temporal profiles they effectuated were very similar, and whose average threshold time was approximately 150 minutes. The standard deviation of the threshold times computed over one hundred realizations were found to be between 1.8 and 7.16 minutes across both models. To see whether a genetic motif in a prokaryotic cell can achieve this degree of precision, we also simulated five variations on the coherent feed-forward motif (CFFM), three of which contained a negative feedback. We found that the performance of these motifs was nowhere near as impressive as the one found in the eukaryotic cell; the best standard deviation was 6.6 minutes. We argue that the significance of these results, the fact and necessity of spatio-temporal precision in the developmental stages of eukaryotes, and the absence of such a precision in prokaryotes, all suggest that the nucleus has evolved, in part, under the selective pressure to achieve highly predictable phenotypes.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate.

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1850-1) contains supplementary material, which is available to authorized users.

The Cooperative Assembly of IFI16 Filaments on dsDNA Provides Insights into Host Defense Strategy

Whether host DNA-receptors have any capacity to distinguish self from nonself at the molecular level is one of the foremost questions in the innate immunity of mammals. By using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve an integral mechanism by which human interferon inducible protein 16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders such as Sjögren's syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16•dsDNA oligomers are clearly different from the previously proposed noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA binding pyrin domain (PYD) of IFI16 that drives the cooperative filament assembly. The surface residues on the PYD that mediate the cooperative DNA-binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling complexes in a switch-like manner, and that it may use the size of naked dsDNA as molecular ruler to distinguish self from nonself.

A switch-like dynamic mechanism for the initiation of replicative senescence

Telomeres are specialized structures protecting chromosomes against genome instability. Telomeres shorten with cell division, and replicative senescence is induced when telomeres are badly eroded. Whereas TRF2 (telomeric-repeat binding factor 2), ATM (ataxia telangiectasia mutated) and p53 have been identified involved in senescence induction, how it is triggered remains unclear. Here, we propose an integrated model associating telomere loss with senescence trigger. We characterize the dynamics of telomere shorting and the p53-centered regulatory network. We show that senescence is initiated in a switch-like manner when both the shortest telomere becomes uncapped and the TRF2-ATM-p53-Siah1 positive feedback loop is switched on. This work provides a coherent picture of senescence induction in terms of telomere shortening and p53 activation.

Asymmetric positive feedback loops reliably control biological responses.

Positive feedback is a common mechanism enabling biological systems to respond to stimuli in a switch-like manner. Such systems are often characterized by the requisite formation of a heterodimer where only one of the pair is subject to feedback. This ASymmetric Self-UpREgulation (ASSURE) motif is central to many biological systems, including cholesterol homeostasis (LXRα/RXRα), adipocyte differentiation (PPARγ/RXRα), development and differentiation (RAR/RXR), myogenesis (MyoD/E12) and cellular antiviral defense (IRF3/IRF7). To understand why this motif is so prevalent, we examined its properties in an evolutionarily conserved transcriptional regulatory network in yeast (Oaf1p/Pip2p). We demonstrate that the asymmetry in positive feedback confers a competitive advantage and allows the system to robustly increase its responsiveness while precisely tuning the response to a consistent level in the presence of varying stimuli. This study reveals evolutionary advantages for the ASSURE motif, and mechanisms for control, that are relevant to pharmacologic intervention and synthetic biology applications.

An Environment-dependent Structural Switch Underlies the Regulation of Carnitine Palmitoyltransferase 1A

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid β-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nβ, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nβ to be noninhibitory, with the relative Nα/Nβ ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nβ ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.