Irreversible disassembly of chiral macrodomains in thylakoids due to photoinhibition

We investigated the effect of photoinhibitory illumination on the chiral macroorganization of the chromophores in spinach thylakoid membranes. By measuring circular dichroism (CD), we found that prolonged (15 min) illumination of membranes with intense white light led to irreversible diminishment of the main CD bands originating from the chiral macroorganization of the chromophores. The irreversible decrease of the main CD bands showed a nearly linear correlation with the extent of photoinhibition which was determined by chlorophyll fluorescence induction. CD measurements also revealed that the excitonic CD bands, which are given rise by short-range interactions between the chromophores inside the complexes or particles, were largely insensitive to the photoinhibitory illumination of the membranes. These data show that, whereas photoinhibitory treatment has no perceptible effect on the molecular architecture of the bulk of the pigment–protein complexes, it leads to a disorganization of their macroarray, and an irreversible disassembly of the chirally organized macrodomains.     

Using optogenetics to tackle systems-level questions of multicellular morphogenesis

Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.     

Protein phosphatase 2A controls the order and dynamics of cell-cycle transitions

Bistability of the Cdk1-Wee1-Cdc25 mitotic control network underlies the switch-like transitions between interphase and mitosis. Here, we show by mathematical modeling and experiments in Xenopus egg extracts that protein phosphatase 2A (PP2A), which can dephosphorylate Cdk1 substrates, is essential for this bistability. PP2A inhibition in early interphase abolishes the switch-like response of the system to Cdk1 activity, promoting mitotic onset even with very low levels of Cyclin, Cdk1, and Cdc25, while simultaneously inhibiting DNA replication. Furthermore, even if replication has already initiated, it cannot continue in mitosis. Exclusivity of S and M phases does not depend on bistability only, since partial PP2A inhibition prevents replication without inducing mitotic onset. In these conditions, interphase-level mitotic kinases inhibit Cyclin E-Cdk2 chromatin loading, blocking initiation complex formation. Therefore, by counteracting both Cdk1 activation and activity of mitotic kinases, PP2A ensures robust separation of S phase and mitosis and dynamic transitions between the two states.     

Irreversible transitions in the 6-phosphofructokinase/fructose 1,6-bisphosphatase cycle.

The dynamics of the fructose 6-phosphate fructose-1,6-bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6-phosphofructokinase and fructose-1,6-bisphosphatase, pyruvate kinase, adenylate kinae and glucose-6-phosphate isomerase were involved. In that multi-enzyme system, the main source of non-linearity is the reciprocal effect of AMP on the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase. Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur. In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions. Two parameters of the system, that is the adenylate energy charge of the influx and the fructose-1,6-bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose-1,6-bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion). This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.     

Back to the future: education for systems-level biologists

We describe a graduate course in quantitative biology that is based on original path-breaking papers in diverse areas of biology; each of these papers depends on quantitative reasoning and theory as well as experiment. Close reading and discussion of these papers allows students with backgrounds in physics, computational sciences or biology to learn essential ideas and to communicate in the languages of disciplines other than their own.     

Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations

The cell-cycle oscillator includes an essential negative-feedback loop: Cdc2 activates the anaphase-promoting complex (APC), which leads to cyclin destruction and Cdc2 inactivation. Under some circumstances, a negative-feedback loop is sufficient to generate sustained oscillations. However, the Cdc2/APC system also includes positive-feedback loops, whose functional importance we now assess. We show that short-circuiting positive feedback makes the oscillations in Cdc2 activity faster, less temporally abrupt, and damped. This compromises the activation of cyclin destruction and interferes with mitotic exit and DNA replication. This work demonstrates a systems-level role for positive-feedback loops in the embryonic cell cycle and provides an example of how oscillations can emerge out of combinations of subcircuits whose individual behaviors are not oscillatory. This work also underscores the fundamental similarity of cell-cycle oscillations in embryos to repetitive action potentials in pacemaker neurons, with both systems relying on a combination of negative and positive-feedback loops.     

Model of calcium oscillations due to negative feedback in olfactory cilia

We present a mathematical model for calcium oscillations in the cilia of olfactory sensory neurons. The underlying mechanism is based on direct negative regulation of cyclic nucleotide-gated channels by calcium/calmodulin and does not require any autocatalysis such as calcium-induced calcium release. The model is in quantitative agreement with available experimental data, both with respect to oscillations and to fast adaptation. We give predictions for the ranges of parameters in which oscillations should be observable. Relevance of the model to calcium oscillations in other systems is discussed.     

Extensive conformational transitions are required to turn on ATP hydrolysis in myosin

Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such “mechanochemical coupling,” especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the “wedge loop.” Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the “wedge loop,” which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 Å away.     

Vicious circles: positive feedback in major evolutionary and ecological transitions

Evolutionary biologists and ecologists often focus on equilibrium states that are subject to forms of negative feedback, such as optima for phenotypic traits or regulation of population sizes. However, recent theoretical and empirical studies show how positive feedback can be instrumental in driving many of the most important and spectacular processes in evolutionary ecology, including the evolution of sex and genetic systems, mating systems, life histories, complex cooperation in insects and humans, ecological specialization, species diversity, species ranges, speciation and extinction. Taken together, this work suggests that positive feedback is more common than is generally appreciated, and that its self-reinforcing dynamics generate the conditions for changes that might otherwise be difficult or impossible for selection or other mechanisms to achieve. Testing for positive feedback requires analysing each causal link in feedback loops, tracking genetic, character and population-dynamic changes across generations, and elucidating the conditions that can result in self-reinforcing change.     

Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell-cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.     

Irreversible Heavy Chain Transfer to Chondroitin

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.     

Connecting cell-cycle activation to neurodegeneration in Drosophila

Studies in cell-culture systems and in postmortem tissue from human disease have suggested a connection between cell-cycle activation and neurodegeneration. The fruit fly Drosophila melanogaster has recently emerged as a powerful model system in which to model neurodegenerative diseases. Here we review work in the fly that has begun to address some of the important questions regarding the relationship between cell-cycle activation and neurodegeneration in vivo, including recent data implicating cell-cycle activation as a downstream effector of tau-induced neurodegeneration. We suggest how powerful research tools in Drosophila might be utilized to approach fundamental questions that remain.     

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.     

Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin

β-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited.     

CD133-positive cells are resistant to TRAIL due to up-regulation of FLIP

Recent research shows that Cancer stem cells (CSCs) are relatively resistant to apoptosis induction. We studied the effect of the immunological apoptogen TRAIL on Jurkat cells enriched in the CD133-positive population. CD133^high Jurkat cells were more resistant to apoptosis than their CD133^low counterparts, and showed higher level of expression of FLIP, an inhibitor of death receptor-mediated apoptosis. Breast cancer MCF7 cells showed high level of expression CD133 in the unseparated culture, with accompanying high level of FLIP. Down-regulation of FLIP by siRNA resulted in sensitisation of the cells to TRAIL, as documented by more robust apoptosis. We conclude that high expression of FLIP is at least one of the reasons for resistance of CSCs to apoptosis induced by the death ligand TRAIL.