The Pierced Lasso Topology Controls Function in Leptin. Journal of Physical Chemistry B 121.4 (2017): 706-718.

Ellinor Haglund, Anna Pilko, Roy Wollman, Patricia Ann Jennings, and Jose Nelson Onuchic

Protein engineering is a powerful tool in drug design and therapeutics, where disulphide bridges are commonly introduced to stabilize proteins. However, these bonds also introduce covalent loops, which are often neglected. These loops may entrap the protein backbone on opposite sides, leading to a "knotted" topology, forming a so-called Pierced Lasso (PL). In this elegant system, the "knot" is held together with a single disulphide bridge where part of the polypeptide chain is threaded through. The size and position of these covalent loops can be manipulated through protein design in vitro, whereas nature uses polymorphism to switch the PL topology. The PL protein leptin shows genetic modification of an N-terminal residue, adding a third cysteine to the same sequence. In an effort to understand the mechanism of threading of these diverse topologies, we designed three loop variants to mimic the polymorphic sequence. This adds elegance to the system under study, as it allows the generation of three possible covalent loops; they are the original wild-type C-terminal loop protein, the fully circularized unthreaded protein, and the N-terminal loop protein, responsible for different lasso topologies. The size of the loop changes the threading mechanism from a slipknotting to a plugging mechanism, with increasing loop size. Interestingly, the ground state of the native protein structure is largely unaffected, but biological assays show that the activity is maximized by properly controlled dynamics in the threaded state. A threaded topology with proper conformational dynamics is important for receptor interaction and activation of the signaling pathways in vivo.

Distinct cellular states determine calcium signaling response. Molecular Systems Biology 12.12 (2016): 894

Jason Yao, Anna Pilko, and Roy Wollman

The heterogeneity in mammalian cells signaling response is largely a result of pre-existing cell-to-cell variability. It is unknown whether cell-to-cell variability rises from biochemical stochastic fluctuations or distinct cellular states. Here, we utilize calcium response to adenosine trisphosphate as a model for investigating the structure of heterogeneity within a population of cells and analyze whether distinct cellular response states coexist. We use a functional definition of cellular state that is based on a mechanistic dynamical systems model of calcium signaling. Using Bayesian parameter inference, we obtain high confidence parameter value distributions for several hundred cells, each fitted individually. Clustering the inferred parameter distributions revealed three major distinct cellular states within the population. The existence of distinct cellular states raises the possibility that the observed variability in response is a result of structured heterogeneity between cells. The inferred parameter distribution predicts, and experiments confirm that variability in IP3R response explains the majority of calcium heterogeneity. Our work shows how mechanistic models and single-cell parameter fitting can uncover hidden population structure and demonstrate the need for parameter inference at the single-cell level.

Signal Transduction at the Single-Cell Level: Approaches to Study the Dynamic Nature of Signaling Networks. Journal of Molecular Biology 428.19 (2016):3669-3682

L Naomi Handly, Jason Yao, and Roy Wollman

Signal transduction, or how cells interpret and react to external events, is a fundamental aspect of cellular function. Traditional study of signal transduction pathways involves mapping cellular signaling pathways at the population level. However, population-averaged readouts do not adequately illuminate the complex dynamics and heterogeneous responses found at the single-cell level. Recent technological advances that observe cellular response, computationally model signaling pathways, and experimentally manipulate cells now enable studying signal transduction at the single-cell level. These studies will enable deeper insights into the dynamic nature of signaling networks.

The Effect of Keystone Individuals on Collective Outcomes Can Be Mediated through Interactions or Behavioral Persistence. The American Naturalist 188.2 (2016)

Noa Pinter-Wollman, Carl N. Keiser, Roy Wollman, and Jonathan N. Pruitt

Collective behavior emerges from interactions among group members who often vary in their behavior. The presence of just one or a few keystone individuals, such as leaders or tutors, may have a large effect on collective outcomes. These individuals can catalyze behavioral changes in other group members, thus altering group composition and collective behavior. The influence of keystone individuals on group function may lead to trade-offs between ecological situations, because the behavioral composition they facilitate may be suitable in one situation but not another. We use computer simulations to examine various mechanisms that allow keystone individuals to exert their influence on group members. We further discuss a trade-off between two potentially conflicting collective outcomes, cooperative prey attack and disease dynamics. Our simulations match empirical data from a social spider system and produce testable predictions for the causes and consequences of the influence of keystone individuals on group composition and collective outcomes. We find that a group’s behavioral composition can be impacted by the keystone individual through changes to interaction patterns or behavioral persistence over time. Group behavioral composition and the mechanisms that drive the distribution of phenotypes influence collective outcomes and lead to trade-offs between disease dynamics and cooperative prey attack.

Paracrine communication maximizes cellular response fidelity in wound signaling. eLife 4 (2015): e09652.

L. Naomi Handly, Anna Pilko, and Roy Wollman

Population averaging due to paracrine communication can arbitrarily reduce cellular response variability. Yet, variability is ubiquitously observed, suggesting limits to paracrine averaging. It remains unclear whether and how biological systems may be affected by such limits of paracrine signaling. To address this question, we quantify the signal and noise of Ca2+ and ERK spatial gradients in response to an in vitro wound within a novel microfluidics-based device. We find that while paracrine communication reduces gradient noise, it also reduces the gradient magnitude. Accordingly we predict the existence of a maximum gradient signal to noise ratio. Direct in vitro measurement of paracrine communication verifies these predictions and reveals that cells utilize optimal levels of paracrine signaling to maximize the accuracy of gradient-based positional information. Our results demonstrate the limits of population averaging and show the inherent tradeoff in utilizing paracrine communication to regulate cellular response fidelity.

Limited specificity of IRF3 and ISGF3 in the transcriptional innate-immune response to double-stranded RNA. Journal of leukocyte biology 98.1 (2015): 119-128.

Diana R. Ourthiague, Harry Birnbaum, Niklas Ortenlöf, Jesse D. Vargas, Roy Wollman, and Alexander Hoffmann

The innate immune response is largely initiated by pathogen-responsive activation of the transcription factor IRF3. Among other target genes, IRF3 controls the expression of IFN-β, which triggers the activation of the transcription factor ISGF3 via the IFNAR. IRF3 and ISGF3 have been reported to control many of the same target genes and together, control the antimicrobial innate-immune program; however, their respective contributions and specificities remain unclear. Here, we used genomic technologies to characterize their specificity in terms of their physical DNA-binding and genetic function. With the use of ChiP-seq and transcriptomic measurements in WT versus ifnar−/− versus ifnar−/−irf3−/− macrophages responding to intracellular dsRNA, we confirmed the known ISGF3 DNA-binding motif and further specified a distinct IRF3 consensus sequence. The functional specificity of IRF3 is particularly pronounced in cytokine/chemokine regulation; yet, even in the control of IFN-β, that specificity is not absolute. By mathematically modeling IFN-β production within an abstracted tissue layer, we find that IRF3 versus ISGF3 specificity may be critical to limiting IFN-β production and ISGF3 activation, temporally and spatially, but that partial overlap in their specificity is tolerable and may enhance the effectiveness of the innate-immune response.

Accurate information transmission through dynamic biochemical signaling networks. Science 346.6215 (2014): 1370-1373.

Jangir Selimkhanov, Brooks Taylor, Jason Yao, Anna Pilko, John Albeck, Alexander Hoffmann, Lev Tsimring, and Roy Wollman

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.

Counting the Ways to Decode Dynamic Signals. Science 343.6177 (2014): 1326-1327.

Roy Wollman

The role of biological signaling networks is to reliably transmit specific information about the extracellular environment to multiple intracellular downstream effectors, allowing the cell to adjust its physiological state to changing conditions. One mechanism that cells use to enhance the performance of signaling networks is the temporal modulation, or dynamics, of the transmitted signals (1–4). The key role that modulating temporal activity of the signal plays in information transmission makes signaling dynamics an attractive target for therapeutic approaches that interfere with the transmission of specific types of information through the network (5). The diversity of temporal modulation strategies seen in various signaling networks suggests that there is no single optimal strategy for making use of dynamic information. Therefore, to uncover the benefits of temporal modulation strategies, it is important to understand how the suitability of each type of signaling dynamics is matched to the nature of the particular information that is being transmitted. Some types of information are transmitted through frequency-modulated signals, whereas other types are transmitted through modulation of signal amplitude or duration. Work by Cai et al. (6) on page 1329 of this issue identifies how the social amoeba Dictyostelium discoideum decodes a temporally dynamic signal to coordinate its development in response to starvation.

Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion. Nature cell biology 14.12 (2012): 1261-1269.

Roy Wollman and Tobias Meyer

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. Cell 149.7 (2012): 1500-1513.

Silvia D.M. Santos, Roy Wollman, Tobias Meyer, and James E. Ferrell Jr.

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.

Cell polarity: quantitative modeling as a tool in cell biology." Science 336.6078 (2012): 175-179.

Mogilner, Alex, Jun Allard, and Roy Wollman

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. Journal of the Royal Society Interface 8.64 (2011): 1562-1573.

Noa Pinter-Wollman, Roy Wollman, Adam Guetz, Susan Holmes, and Deborah M. Gordon

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 ant's 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.

A genome-wide siRNA screen reveals various modulators of the IRE1-XBP-1 signaling branch of the unfolded protein response. Cancer Research 71.8 Supplement (2011): 2068-2068.

Jing Zhang, Alice Banh, Priya Jayachandran, Roy Wollman, David E. Solow-Codero, Tobias Meyer, Quynh-Thu Le, and Albert C. Koong

The unfolded protein response (UPR) is an essential cellular mechanism orchestrating endoplasmic reticulum (ER) homeostasis under various cytotoxic stresses. Dysregulation of the UPR signaling pathways is linked to multiple human diseases, including cancer. The inositol requiring kinase 1 (IRE1)-X-box binding protein 1 (XBP-1) pathway is the most evolutionarily conserved signaling branch of the UPR. When activated, the endoribonuclease activity of the IRE1 cleaves the XBP-1 mRNA and generates a spliced form of XBP-1 which transcriptionally regulates cell metabolism, proliferation, survival and apoptosis. Here, we employed a genome-wide siRNA screen to systematically identify genes modulating the IRE1-XBP-1 signaling pathway of the UPR. We induced cellular ER stress with bortezomib, a proteasome inhibitor, and monitored the expression of XBP-1-luciferase fusion protein in which luciferase is fused in-frame with the spliced form of XBP-1. We identified over 100 of genes whose downregulation results in inhibition of bortezomib-induced XBP-1 splicing. These genes include diverse subsets of proteins that are involved in mRNA processing, transcription, cell cycle regulation and cell proliferation and differentiation. Furthermore, the screen reveals a connection of several oncogenes and tumor suppressors, such as Myc, Akt and cyclin K, to the UPR, underlining the important role of the IRE1-XBP-1 signaling branch in human cancers. In summary, our data suggest that the UPR is tightly connected with previously unappreciated pathways regulating various cellular processes.

Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope. The Journal of cell biology 188.1 (2010): 49-68.

Gul Civelekoglu-Scholey, Li Tao, Ingrid Brust-Mascher, Roy Wollman, and Jonathan M. Scholey

We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator.

Computer simulations predict that chromosome movements and rotations accelerate mitotic spindle assembly without compromising accuracy. Proceedings of the National Academy of Sciences 106.37 (2009): 15708-15713."]

Raja Paul, Roy Wollman, William T. Silkworth, Isaac K. Nardi, Daniela Cimini, and Alex Mogilner

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.

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Molecular cell 35.2 (2009): 228-239.

Renee D. Paulsen, Deena V. Soni, Roy Wollman, Angela T. Hahn, Muh-Ching Yee, Anna Guan, Jayne A. Hesley, Steven C. Miller, Evan F. Cromwell, David E. Solow-Cordero, Tobias Meyer, and Karlene A. Cimprich

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 (γH2AX) 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 γH2AX 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.

Reverse engineering of force integration during mitosis in the Drosophila embryo. Molecular systems biology 4.1 (2008).

Roy Wollman, Gul Civelekoglu-Scholey, Jonathan M Scholey, and Alex Mogilner

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.

High throughput microscopy: from raw images to discoveries. Journal of cell science 120.21 (2007): 3715-3722.

Roy Wollman and Nico Stuurman

Technological advances in automated microscopy now allow rapid acquisition of many images without human intervention, images that can be used for large-scale screens. The main challenge in such screens is the conversion of the raw images into interpretable information and hence discoveries. This post-acquisition component of image-based screens requires computational steps to identify cells, choose the cells of interest, assess their phenotype, and identify statistically significant `hits'. Designing such an analysis pipeline requires careful consideration of the necessary hardware and software components, image analysis, statistical analysis and data presentation tools. Given the increasing availability of such hardware and software, these types of experiments have come within the reach of individual labs, heralding many interesting new ways of acquiring biological knowledge.

Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316.5823 (2007): 417-421.

Gohta Goshima, Roy Wollman, Sarah S. Goodwin, Nan Zhang, Jonathan M. Scholey, Ronald D. Vale1, and Nico Stuurman

The formation of a metaphase spindle, a bipolar microtubule array with centrally aligned chromosomes, is a prerequisite for the faithful segregation of a cell's genetic material. Using a full-genome RNA interference screen of Drosophila S2 cells, we identified about 200 genes that contribute to spindle assembly, more than half of which were unexpected. The screen, in combination with a variety of secondary assays, led to new insights into how spindle microtubules are generated; how centrosomes are positioned; and how centrioles, centrosomes, and kinetochores are assembled.

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays. Current biology 16.23 (2006): 2293-2302.

Li Tao, Alex Mogilner, Gul Civelekoglu-Scholey, Roy Wollman, James Evans, Henning Stahlberg, and Jonathan M. Scholey

Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos.

Quantitative modeling in cell biology: what is it good for?. Developmental cell 11.3 (2006): 279-287.

Alex Mogilner, Roy Wollman, and Wallace F. Marshall

Recently, there has been a surge in the number of pioneering studies combining experiments with quantitative modeling to explain both relatively simple modules of molecular machinery of the cell and to achieve system-level understanding of cellular networks. Here we discuss the utility and methods of modeling and review several current models of cell signaling, cytoskeletal self-organization, nuclear transport, and the cell cycle. We discuss successes of and barriers to modeling in cell biology and its future directions, and we argue, using the field of bacterial chemotaxis as an example, that the closer the complete systematic understanding of cell behavior is, the more important modeling becomes and the more experiment and theory merge.

Modeling mitosis. Trends in cell biology 16.2 (2006): 88-96.

Alex Mogilner, Roy Wollman, Gul Civelekoglu-Scholey, and Jonathan Scholey

The mitotic spindle is a fascinating protein machine that uses bipolar arrays of dynamic microtubules and many mitotic motors to coordinate the accurate segregation of sister chromatids. Here we discuss recent mathematical models and computer simulations that, in concert with experimental studies, help explain the molecular mechanisms by which the spindle machinery performs its crucial functions. We review current models of spindle assembly, positioning, maintenance and elongation; of chromosome capture and congression; and of the spindle assembly checkpoint. We discuss some limitations of the application of modeling to other aspects of mitosis and the feasibility of building more comprehensive system-level models.[/expand] 

Length control of the metaphase spindle. Current Biology 15.22 (2005): 1979-1988.

Gohta Goshima, Roy Wollman, Nico Stuurman, Jonathan M. Scholey, and Ronald D. Vale

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.

APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Molecular biology of the cell 16.10 (2005): 4609-4622.

Rebecca A. Green, Roy Wollman, and Kenneth B. Kaplan

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC1–1450) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC1–1450 acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC1–1450 compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

QuasiMotiFinder: protein annotation by searching for evolutionarily conserved motif-like patterns. Nucleic acids research (2005): W255-W261.

Roee Gutman, Carine Berezin, Roy Wollman, Yossi Rosenberg, and Nir Ben-Tal

Sequence signature databases such as PROSITE, which include amino acid segments that are indicative of a protein's function, are useful for protein annotation. Lamentably, the annotation is not always accurate. A signature may be falsely detected in a protein that does not carry out the associated function (false positive prediction, FP) or may be overlooked in a protein that does carry out the function (false negative prediction, FN). A new approach has emerged in which a signature is replaced with a sequence profile, calculated based on multiple sequence alignment (MSA) of homologous proteins that share the same function. This approach, which is superior to the simple pattern search, essentially searches with the sequence of the query protein against an MSA library. We suggest here an alternative approach, implemented in the QuasiMotiFinder web server (http://quasimotifinder.tau.ac.il/), which is based on a search with an MSA of homologous query proteins against the original PROSITE signatures. The explicit use of the average evolutionary conservation of the signature in the query proteins significantly reduces the rate of FP prediction compared with the simple pattern search. QuasiMotiFinder also has a reduced rate of FN prediction compared with simple pattern searches, since the traditional search for precise signatures has been replaced by a permissive search for signature-like patterns that are physicochemically similar to known signatures. Overall, QuasiMotiFinder and the profile search are comparable to each other in terms of performance. They are also complementary to each other in that signatures that are falsely detected in (or overlooked by) one may be correctly detected by the other.

Efficient chromosome capture requires a bias in the ‘search-and-capture’process during mitotic-spindle assembly. Current Biology 15.9 (2005): 828-832.

Roy Wollman, Eric N Cytrynbaum, Joshua T. Jones, Tobias Meyer, Jonathan M. Scholey, and Alex Mogilner

The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is “search-and-capture,” in which dynamically unstable microtubules (MTs) search space to capture chromosomes [1]. Although existing theoretical estimates [2 and 3] suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor [4, 5 and 6] that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.