Binary Switch Research Articles

Tpz1 controls a telomerase-nonextendible telomeric state and coordinates switching to an extendible state via Ccq1

Telomeres are nucleoprotein complexes comprising telomeric DNA repeats bound by the multiprotein shelterin complex. A dynamic binary switch between telomerase-extendible and telomerase-nonextendible telomeric states determines telomere length homeostasis. However, the molecular nature of the nonextendible state is largely unknown. Here, we show that, in fission yeast, Tpz1 (the ortholog of human TPP1)-mediated complete linkage within the shelterin complex, bridging telomeric dsDNA to ssDNA, controls the telomerase-nonextendible state. Disruption of this linkage leads to unregulated telomere elongation while still retaining the shelterin components on telomeres. Therefore, the linkage within the shelterin components, rather than the individual shelterin components per se, defines the telomerase-nonextendible state. Furthermore, epistasis analyses reveal that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Our results suggest critical regulatory roles of Tpz1 in the telomere binary switch.

Regulating DNA Replication in Eukarya

DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.

The dark side of protein kinases: FRET toolbox illuminates a hidden conformation in PKC catalysis

Kinases have been traditionally viewed as binary switches that are activated or inactivated by varied small molecule second messengers, post‐translational modifications, and allosteric regulatory events. However, an emerging view is that kinases function in a fashion analogous to micro‐processors, coupling varied upstream signals to specific downstream effects. Despite the importance of kinases in myriad signaling pathways and processes, contemporary methods and techniques do not allow for in‐depth study of spatial and temporal co‐ordination of the intra‐ and inter‐molecular interactions that regulate kinase function in cells. Here we present a new FRET‐based approach termed the kinase toolbox that was used to dissect the mechanisms of activation and auto‐regulation of protein kinase C (PKC). Utilizing several FRET sensors embedded into PKCα our studies resolved a second active conformation required for catalysis. The nucleotide binding pocket is allosterically linked with the c‐terminal tail of the catalytic domain and binding of ATP or ADP differentially stabilizes this conformation. Additional stabilization of this second state through inter‐molecular interactions modulates kinase activity by enhancing nucleotide exchange, ascribing a necessary function to the new state. The development of the kinase toolbox outlines how similar mechanistic insights may be gleaned from other AGC kinases.

Two modes of integrin activation form a binary molecular switch in adhesion maturation.

Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin-integrin interactions--vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)--in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.

Transforming mutations of RAC guanosine triphosphatases in human cancers

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.

Chapter Six - Development of EHop-016: A Small Molecule Inhibitor of Rac

The Rac inhibitor EHop-016 was developed as a compound with the potential to inhibit cancer metastasis. Inhibition of the first step of metastasis, migration, is an important strategy for metastasis prevention. The small GTPase Rac acts as a pivotal binary switch that is turned "on" by guanine nucleotide exchange factors (GEFs) via a myriad of cell surface receptors, to regulate cancer cell migration, survival, and proliferation. Unlike the related GTPase Ras, Racs are not usually mutated, but overexpressed or overactivated in cancer. Therefore, a rational Rac inhibitor should block the activation of Rac by its upstream effectors, GEFs, and the Rac inhibitor NSC23766 was developed using this rationale. However, this compound is ineffective at inhibiting the elevated Rac activity of metastatic breast cancer cells. Therefore, a panel of small molecule compounds were derived from NSC23766 and screened for Rac activity inhibition in metastatic cancer cells. EHop-016 was identified as a compound that blocks the interaction of Rac with the GEF Vav in metastatic human breast cancer cells with an IC50 of ~1μM. At higher concentrations (10μM), EHop-016 inhibits the related Rho GTPase Cdc42, but not Rho, and also reduces cell viability. Moreover, EHop-016 inhibits the activation of the Rac downstream effector p21-activated kinase, extension of motile actin-based structures, and cell migration. Future goals are to develop EHop-016 as a therapeutic to inhibit cancer metastasis, either individually or in combination with current anticancer compounds. The next generation of EHop-016-based Rac inhibitors is also being developed.

Output Feedback Control of Discrete Impulsive Switched Systems with State Delays and Missing Measurements

This paper is concerned with the problem of dynamic output feedback (DOF) control for a class of uncertain discrete impulsive switched systems with state delays and missing measurements. The missing measurements are modeled as a binary switch sequence specified by a conditional probability distribution. The problem addressed is to design an output feedback controller such that for all admissible uncertainties, the closed-loop system is exponentially stable in mean square sense. By using the average dwell time approach and the piecewise Lyapunov function technique, some sufficient conditions for the existence of a desired DOF controller are derived, then an explicit expression of the desired controller is given. Finally, a numerical example is given to illustrate the effectiveness of the proposed method.

Single Neuronal Snare Complexes Zipper in Three Distinct Stages

SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here we used optical tweezers to observe in real time a long-sought SNARE assembly intermediate in which only the membrane-distal N-terminal half of the bundle is assembled. Our finding supports the zippering hypothesis, but suggests that zippering proceeds through three sequential binary switches, not continuously, in the N- and C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force which mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion.

Huffman scanning: Using language models within fixed-grid keyboard emulation

Individuals with severe motor impairments commonly enter text using a single binary switch and symbol scanning methods. We present a new scanning method – Huffman scanning – which uses Huffman coding to select the symbols to highlight during scanning, thus minimizing the expected bits per symbol. With our method, the user can select the intended symbol even after switch activation errors. We describe two varieties of Huffman scanning – synchronous and asynchronous – and present experimental results, demonstrating speedups over row/column and linear scanning.

Information Processing with Electron Spins

Information processors process information in a variety of ways. The human brain processes information through a highly interconnected system of neurons and synapses, while a digital computer processes information by having a binary switch toggle on and off in response to a stream of binary bits. The “switch” is the most primitive unit of the modern computer. The better it is (faster, more energy efficient, more reliable, etc.), the more advanced is the computer hardware. Energy efficiency, however, is more important than any other attribute, not so much because energy is costly, but because too much energy dissipation prevents increasing the density of switches on a chip that is necessary to make the chip increasingly more powerful. Reducing dissipation entails radically new and often revolutionary approaches for implementing the switch. One such approach is to encode digital bit information in the spin polarization of a single electron (or ensemble of electrons) and then using two mutually antiparallel polarizations to represent the binary bits 0 and 1. Switching between the bits can be accomplished by simply flipping the polarizations of the spins, which takes very little energy. Such switches are extremely energy efficient if designed properly, but they are somewhat slower than traditional transistor-based switches and can be more error prone. This paper discusses the pros and cons of spin-based switches and introduces the reader to the most recent advancements in information processing predicated on encoding information in electron spin polarization.

Phosphoinositide 5-phosphatases: How do they affect tumourigenesis?

The activity of biological molecules is often affected by their phosphorylation state. Regulatory phosphorylation operates as a binary switch and is usually controlled by counteracting kinases and phosphatases. However, phosphatidylinositol (PtdIns) has three phosphorylation sites on its inositol ring. The phosphorylation status of PtdIns is controlled by multiple kinases and phosphatases with distinct substrate specificities, serving as a 'lipid code' or 'phosphoinositide code'. Class I phosphoinositide 3-kinase (PI3K) converts PtdIns(4,5)P₂ to PtdIns(3,4,5)P₃, which plays a pivotal role in signals controlling glucose uptake, cytoskeletal reorganization, cell proliferation and apoptosis. PI3K is pro-oncogenic, whereas phosphoinositide phosphatases that degrade PtdIns(3,4,5)P₃ are not always anti-oncogenic. Recent studies have revealed the unique characteristics of phosphoinositide 5-phosphatases.

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

SummaryNeuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

PIDD Death-Domain Phosphorylation by ATM Controls Prodeath versus Prosurvival PIDDosome Signaling

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.

Advantages and limitations of cell-based assays for GTPase activation and regulation

Small GTPases of the Ras superfamily are important regulators of many cellular functions, including signal transduction, cytoskeleton assembly, metabolic regulation, organelle biogenesis and intracellular transport. Most GTPases act as binary switches, being “on” in the active, GTP-bound state and “off” in the inactive, GDP-bound state, and cycle between the two states with the aid of accessory proteins, referred to as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). This review will focus on the ADP-ribosylation factors (Arfs), a family of G-proteins that are essential regulators of carrier vesicle formation during vesicular transport. As for most other GTPases, the Arfs themselves are vastly outnumbered by the proteins that regulate them, and a major focus in the field has been to define the functional relationships between individual GEFs and GAPs and their substrates at the cellular level. Over the years, a variety of methods have been developed to measure GTPase activation in vitro and in vivo. In vitro analysis will be discussed in the accompanying article by Randazzo and colleagues. Here we will focus on cell- and tissue-based assays and their advantages/disadvantages relative to cell-free systems.

Modeling cuttlefish behavioural chromatophore response

We build a model of cuttlefish chromatophore excitation patterns due to environmental input. Specific inputs generate scripted chromtophore pattern responses on the surface of the skin. More complicated responses are then assembled from these templates as sequences of inputs arrive. We build a simplistic cuttlefish brain using neural architecture modeling tools written in MatLab which allow us to construct the brain model from individual modules using a vector addressing scheme [1] (Figure ​(Figure11). Figure 1 Cuttlefish chromatophore processing Inputs come into the sensory input module and are processed by the cuttlefish brain architecture into signals sent to the output module. The signals activate the individual chromatophores in the usual way giving an essentially binary on/ off pixel response. Different cues in the environment are mapped to known cuttlefish pigmentation overlays on the surface of the skin. Individual simple cues result in coarse patterns of chromotophore activation and upon receiving a sequence of simple cues, more complicated responses are constructed in a hierarchical fashion [1]. The chromatophore visibility is known to be due to a brain signal which when received energizes a ring of muscle which contracts and pushes a dot of ink up to the surface of the skin. Hence, activation signals lead to visible dots and lack of activation signal can be inferred from the loss of the pigment dot. Our model therefore treats each chromatophore as a binary switch which moves from 1 to 0 or vice versa depending on the signal sent to the output module from our cuttlefish brain processing [3,4].

Kinetic and cell-based analyses of GTPase regulators

Regulatory GTPases are often portrayed as binary molecular switches that control a wide range of cellular processes, including, but not limited to, the generation of second messengers (e.g., cAMP and inositol phosphates), intracellular traffic, cytoskeleton organization and cell proliferation. GEF stimulators and GAP inhibitors regulate the nucleotide-bound state of these proteins. Because of the relevance of GTPases and their regulators to human diseases, they comprise a major therapeutic target. Currently, most of the information about GTPase regulators comes from structure analyses. Such structural information is limited to certain conditions and does not necessarily reflect specificity or physiological activity. To address questions about specificity and mechanisms of action, kinetic and cell-based analyses of GTPase regulators is necessary. Here, we compare these two approaches in the context of regulators of Arf and heterotrimeric G-proteins.

Sharing Control With Haptics

Haptic shared control was investigated as a human-machine interface that can intuitively share control between drivers and an automatic controller for curve negotiation. As long as automation systems are not fully reliable, a role remains for the driver to be vigilant to the system and the environment to catch any automation errors. The conventional binary switches between supervisory and manual control has many known issues, and haptic shared control is a promising alternative. A total of 42 respondents of varying age and driving experience participated in a driving experiment in a fixed-base simulator, in which curve negotiation behavior during shared control was compared to during manual control, as well as to three haptic tunings of an automatic controller without driver intervention. Under the experimental conditions studied, the main beneficial effect of haptic shared control compared to manual control was that less control activity (16% in steering wheel reversal rate, 15% in standard deviation of steering wheel angle) was needed for realizing an improved safety performance (e.g., 11% in peak lateral error). Full automation removed the need for any human control activity and improved safety performance (e.g., 35% in peak lateral error) but put the human in a supervisory position. Haptic shared control kept the driver in the loop, with enhanced performance at reduced control activity, mitigating the known issues that plague full automation. Haptic support for vehicular control ultimately seeks to intuitively combine human intelligence and creativity with the benefits of automation systems.

Abstract 1433: TC21/R-Ras2 is a critical mediator of the Nf1 oncogenic switch

Abstract The TC21/R-Ras2 protein is an oncogenic member of the Ras family. Ras proteins act as binary switches which are ‘active’ when bound to GTP and ‘inactive’ when bound to GDP. GTPase activating proteins (GAPs) accelerate the hydrolysis from Ras-GTP to Ras-GDP. This Ras-related GTPase TC21 has transforming capabilities similar to H, N and K-Ras. Activated alleles of TC21 transform epithelial and fibroblast cell lines and induce tumors in vivo. To explore the role of TC21 in cancer we used a model of Ras activation driven by loss of the NF1 tumor suppressor protein, a GAP for all Ras proteins including TC21. Thus loss of NF1 predicts increased levels of activated TC21/R-Ras2. NF1 loss in Schwann cells of the peripheral nervous system causes benign tumors known as neurofibromas. Neurofibromas can transform to sarcomas known as malignant peripheral nerve sheath tumors (MPNSTs). We found that TC21 loss delayed benign neurofibroma formation in Nf1fl/fl;DhhCre mice. Nf1 loss increased mRNA encoding the cytokine transforming growth factor-beta (TGF-β) and rendered Schwann cell progenitors insensitive to TGF-β; these phenotypes could be rescued by the Ras-related protein TC21/R-Ras2 and were mediated through TGF-β receptors. Conversely, growth of Nf1;Trp53 brain tumors and NF1−/− MPNST sarcomas were accelerated by TC21 loss. MPNST from Nf1;Trp53 mice and NF1−/− MPNST xenografts had increased levels of mRNA encoding TGF- ≤ ligands, and blocking TGF- ≤ decreased sarcoma size induced by shTC21. In benign and malignant tumors an AKT-dependent pathway regulated TGF- ≤ expression. Indicating relevance to human tumorigenesis, global gene expression analyses demonstrated increases in expression of TGF- ≤ ligands and decreases in TGF- ≤ receptors in human neurofibromas and MPNSTs. Thus, we identify critical roles for TC21 in regulation of TGF- ≤ expression. The results are important because TGF- ≤ acts as a tumor suppressor in numerous types of benign tumors and is also known for its oncogenic role in cellular transformation and tumor progression. While it has been known that Ras proteins are involved in TGF- ≤ mediated tumor suppression and oncogenesis, integration between the pathways is incompletely understood, especially in vivo. The study demonstrates that TC21 can be a major regulator of the duality of TGF- ≤ effects on tumorigenesis in vivo. This work was supported by a grant to NR (NIH P50NS057531-03). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1433. doi:1538-7445.AM2012-1433

Abstract 4166: Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

Abstract We report that ERK/MAPK signaling mediates tumor suppression. Oncogenic forms of ras are found in approximately 25% of all human cancers and are generally associated with uncontrolled proliferation of cancer cells. However, expression of oncogenic ras in normal cells is associated with cellular senescence. In primary cells expressing oncogenic ras, ERK knockdown by shRNA prevented the activation of the DNA damage response, p53, p16INK4a/Rb and senescence and cooperated with hTERT in transformation. In human prostate neoplasms, high levels of phosphorylated-ERK were found in benign lesions while in malignant lesions lower levels correlated with a higher Gleason score and earlier disease relapse after treatment. Mechanistically, the antitumor effects of ERK/MAPK signaling included the selective proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions and cell signaling and the inhibition of the phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling pathway. We thus show for the first time that the outcome of ERK/MAPK signaling can be controlled at the level of ERK1/2, the final members of the pathway. A quantitative control at the end of the pathway provides a mechanism for specific modulation of ERK/MAPK signaling since controlling the input may also affect the numerous signaling pathways activated by membrane receptors. The data also indicate that the levels of the ERK kinases control a binary switch between cell proliferation and senescence and explain the maintenance of the senescence phenotype despite the presence of constitutively activated growth factor signaling pathways. Since inhibitors of the ERK pathway are under consideration for clinical use, we caution that they may trigger the escape of dormant senescent cells leading to malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4166. doi:1538-7445.AM2012-4166