Roy Wollman

A Genome-wide siRNA Screen Reveals Diverse Cellular Processes and Pathways that Mediate Genome Stability

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Length Control of the Metaphase Spindle

BACKGROUND The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.

Accurate information transmission through dynamic biochemical signaling networks

Stochasticity inherent to biochemical reactions (intrinsic noise) and variability in cellular states (extrinsic noise) degrade information transmitted through signaling networks. We analyzed the ability of temporal signal modulation—that is, dynamics—to reduce noise-induced information loss. In the extracellular signal–regulated kinase (ERK), calcium (Ca2+), and nuclear factor kappa-B (NF-κB) pathways, response dynamics resulted in significantly greater information transmission capacities compared to nondynamic responses. Theoretical analysis demonstrated that signaling dynamics has a key role in overcoming extrinsic noise. Experimental measurements of information transmission in the ERK network under varying signal-to-noise levels confirmed our predictions and showed that signaling dynamics mitigate, and can potentially eliminate, extrinsic noise–induced information loss. By curbing the information-degrading effects of cell-to-cell variability, dynamic responses substantially increase the accuracy of biochemical signaling networks. Signaling dynamics increase the information transmission capacity of biochemical signaling pathways. Dynamic signals enhance information transfer Cells need to process information about their external environment reliably to survive. However, variation, or noise, in biochemical reactions, or in the states of individual cells, make it hard for a cell to detect concentration, rather than just the presence or absence of an activating ligand. Selimkhanov et al. show that cellular signaling circuits get around this problem by continually monitoring signals over time. Such dynamic responses in cultured human cells more effectively distinguish signals from noise and thus avoid loss of information transmitted to the cell from external signals. Science, this issue p. 1370

Cell Polarity: Quantitative Modeling as a Tool in Cell Biology

Among a number of innovative approaches that have modernized cell biology, modeling has a prominent yet unusual place. One popular view is that we progress linearly, from conceptual to ever more detailed models. We review recent discoveries of cell polarity mechanisms, in which modeling played an important role, to demonstrate that the experiment-theory feedback loop requires diverse models characterized by varying levels of biological detail and mathematical complexity. We argue that a quantitative model is a tool that has to fit an experimental study, and the model’s value should be judged not by how complex and detailed it is, but by what could be learned from it.

The effect of individual variation on the structure and function of interaction networks in harvester ants.

Social insects exhibit coordinated behaviour without central control. Local interactions among individuals determine their behaviour and regulate the activity of the colony. Harvester ants are recruited for outside work, using networks of brief antennal contacts, in the nest chamber closest to the nest exit: the entrance chamber. Here, we combine empirical observations, image analysis and computer simulations to investigate the structure and function of the interaction network in the entrance chamber. Ant interactions were distributed heterogeneously in the chamber, with an interaction hot-spot at the entrance leading further into the nest. The distribution of the total interactions per ant followed a right-skewed distribution, indicating the presence of highly connected individuals. Numbers of ant encounters observed positively correlated with the duration of observation. Individuals varied in interaction frequency, even after accounting for the duration of observation. An ants interaction frequency was explained by its path shape and location within the entrance chamber. Computer simulations demonstrate that variation among individuals in connectivity accelerates information flow to an extent equivalent to an increase in the total number of interactions. Individual variation in connectivity, arising from variation among ants in location and spatial behaviour, creates interaction centres, which may expedite information flow.

Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion

The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca2+ and PtdIns(4,5)P2 lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca2+ level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca2+ levels subsequently increase. Thus, cells employ oscillating levels of Ca2+, PtdIns(4,5)P2 and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

Spatial positive feedback at the onset of mitosis.

Mitosis is triggered by the activation of Cdk1-cyclin B1 and its translocation from the cytoplasm to the nucleus. Positive feedback loops regulate the activation of Cdk1-cyclin B1 and help make the process irreversible and all-or-none in character. Here we examine whether an analogous process, spatial positive feedback, regulates Cdk1-cyclin B1 redistribution. We used chemical biology approaches and live-cell microscopy to show that nuclear Cdk1-cyclin B1 promotes the translocation of Cdk1-cyclin B1 to the nucleus. Mechanistic studies suggest that cyclin B1 phosphorylation promotes nuclear translocation and, conversely, nuclear translocation promotes cyclin B1 phosphorylation, accounting for the feedback. Interfering with the abruptness of Cdk1-cyclin B1 translocation affects the timing and synchronicity of subsequent mitotic events, underscoring the functional importance of this feedback. We propose that spatial positive feedback ensures a rapid, complete, robust, and irreversible transition from interphase to mitosis and suggest that bistable spatiotemporal switches may be widespread in biological regulation.

Computer simulations predict that chromosome movements and rotations accelerate mitotic spindle assembly without compromising accuracy

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10–20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.

Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope

The lamin-B nuclear envelope stabilizes spindle microtubules by keeping the competitive motility of opposing-force kinesins in check.

Reverse engineering of force integration during mitosis in the Drosophila embryo

The mitotic spindle is a complex macromolecular machine that coordinates accurate chromosome segregation. The spindle accomplishes its function using forces generated by microtubules (MTs) and multiple molecular motors, but how these forces are integrated remains unclear, since the temporal activation profiles and the mechanical characteristics of the relevant motors are largely unknown. Here, we developed a computational search algorithm that uses experimental measurements to ‘reverse engineer’ molecular mechanical machines. Our algorithm uses measurements of length time series for wild‐type and experimentally perturbed spindles to identify mechanistic models for coordination of the mitotic force generators in Drosophila embryo spindles. The search eliminated thousands of possible models and identified six distinct strategies for MT–motor integration that agree with available data. Many features of these six predicted strategies are conserved, including a persistent kinesin‐5‐driven sliding filament mechanism combined with the anaphase B‐specific inhibition of a kinesin‐13 MT depolymerase on spindle poles. Such conserved features allow predictions of force–velocity characteristics and activation–deactivation profiles of key mitotic motors. Identified differences among the six predicted strategies regarding the mechanisms of prometaphase and anaphase spindle elongation suggest future experiments.