Life Sciences Research for Lifelong Health

Publications karen-lipkow

Title / Authors / Details Open Access Download

Higher order assembly: folding the chromosome.
Sewitz SA, Fahmi Z, Lipkow K

The linear molecules of DNA that constitute a eukaryotic genome have to be carefully organised within the nucleus to be able to correctly direct gene expression. Microscopy and chromosome capture methods have revealed a hierarchical organisation into territories, domains and subdomains that ensure the accessibility of expressed genes and eventually chromatin loops that serve to bring gene enhancers into proximity of their target promoters. A rapidly growing number of genome-wide datasets and their analyses have given detailed information into the conformation of the entire genome, allowing evolutionary insights, observations of genome rearrangements during development and the identification of new gene-to-disease associations. The field is now progressing into using computational models of genome dynamics to investigate the mechanisms that shape genome structure, placing increasing importance on the role of chromatin associated proteins for this process.

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Current opinion in structural biology, 42, 1879-033X, 162-168, 2017

PMID: 28284913

Open Access

Systems Biology Approaches for Understanding Genome Architecture.
Sewitz S, Lipkow K

The linear and three-dimensional arrangement and composition of chromatin in eukaryotic genomes underlies the mechanisms directing gene regulation. Understanding this organization requires the integration of many data types and experimental results. Here we describe the approach of integrating genome-wide protein-DNA binding data to determine chromatin states. To investigate spatial aspects of genome organization, we present a detailed description of how to run stochastic simulations of protein movements within a simulated nucleus in 3D. This systems level approach enables the development of novel questions aimed at understanding the basic mechanisms that regulate genome dynamics.

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Methods in molecular biology (Clifton, N.J.), 1431, 1940-6029, 109-26, 2016

PMID: 27283305

An integrated model of transcription factor diffusion shows the importance of intersegmental transfer and quaternary protein structure for target site finding.
Schmidt HG, Sewitz S, Andrews SS, Lipkow K

We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs) around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a 'hopping' motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure.

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PloS one, 9, 1932-6203, e108575, 2014

PMID: 25333780

Open Access

The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family, defined by the adjacent, region-specific homeotic gene spalt.
Barrio R, Shea MJ, Carulli J, Lipkow K, Gaul U, Frommer G, Schuh R, Jäckle H, Kafatos FC

We report the full coding sequence of a new Drosophila gene, spalt-related, which is homologous and adjacent to the region-specific homeotic gene, spalt. Both genes have three widely spaced sets of C2H2 zinc finger motifs, but spalt-related encodes a fourth pair of C-terminal fingers resembling the Xenopus homologue, Xsal-1. The degrees of sequence divergence among all three members of this family are comparable, suggesting that the Drosophila genes originated from an ancient gene duplication. The spalt-related gene is expressed with quantitative variations from mid-embryogenesis (8-12 h) to the adult stage, but not in ovaries or early embryos. Expression is localized to limited parts of the body, including specific cell populations in the nervous system. In the wing disc, spalt and spalt-related are expressed in indistinguishable domains; in the nervous system and some other organs the expression patterns extensively overlap but are not identical, indicating that the genes have partially diverged in terms of developmental regulation. A characteristic central set of zinc fingers specifically binds to an A/T-rich consensus sequence, defining some DNA binding properties of this ancient family of nuclear factors.

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Development genes and evolution, 206, 0949-944X, 315-25, 1996

PMID: 24173589

The autoregulation of a eukaryotic DNA transposon.
C Claeys Bouuaert, K Lipkow, SS Andrews, D Liu, R Chalmers

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable. DOI:http://dx.doi.org/10.7554/eLife.00668.001.

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eLife, 2, , e00668, 2013

PMID: 23795293
DOI: 10.7554/eLife.00668

Open Access

Model for Protein Concentration Gradients in the Cytoplasm.
K Lipkow, DJ Odde

Intracellular protein concentration gradients are generally thought to be unsustainable at steady-state due to diffusion. Here we show how protein concentration gradients can theoretically be sustained indefinitely through a relatively simple mechanism that couples diffusion to a spatially segregated kinase-phosphatase system. Although it is appreciated that such systems can theoretically give rise to phosphostate gradients, it has been assumed that they do not give rise to gradients in the total protein concentration. Here we show that this assumption does not hold if the two forms of protein have different diffusion coefficients. If, for example, the phosphorylated state binds selectively to a second larger protein or protein complex then a steady state gradient in total protein concentration will be created. We illustrate the principle with an analytical solution to the diffusion-reaction problem and by stochastic individual-based simulations using the Smoldyn program. We argue that protein gradients created in this way need to be considered in experiments using fluorescent probes and could in principle encode spatial information in the cytoplasm.

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Cellular and molecular bioengineering, 1, 1, 84-92, 2008

PMID: 21152415
DOI: 10.1007/s12195-008-0008-8

Open Access

Introducing simulated cellular architecture to the quantitative analysis of fluorescent microscopy.
MA DePristo, L Chang, RD Vale, SM Khan, K Lipkow

Biological cells are complex and highly dynamic: many macromolecules are organized in loose assemblies, clusters or highly structured complexes, others exist most of the time as freely diffusing monomers. They move between regions and compartments through diffusion and enzyme-mediated transport, within a heavily crowded cytoplasm. To make sense of this complexity, computational models, and, in turn, quantitative in vivo data are needed. An array of fluorescent microscopy methods is available, but due to the inherent noise and complexity inside the cell, they are often hard to interpret. Using the example of fluorescence recovery after photobleaching (FRAP) and the bacterial chemotaxis system, we are here introducing detailed spatial simulations as a new approach in analysing such data.

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Progress in biophysics and molecular biology, 100, 1-3, 25-32, 2009

PMID: 19628003
DOI: 10.1016/j.pbiomolbio.2009.07.002

Open Access

Amelioration of protein misfolding disease by rapamycin: translation or autophagy?
A Wyttenbach, S Hands, MA King, K Lipkow, AM Tolkovsky

Rapamycin is an inhibitor of mTOR, a key component of the mTORC1 complex that controls the growth and survival of cells in response to growth factors, nutrients, energy balance and stresses. The downstream targets of mTORC1 include ribosome biogenesis, transcription, translation and macroautophagy. Recently it was proposed that rapamycin and its derivatives enhance the clearance (and/or reduce the accumulation) of mutant intracellular proteins causing proteinopathies such as tau, alpha-synuclein, ataxin-3, and full-length or fragments of huntingtin containing a polyglutamine (polyQ) expansion, by upregulating macroautophagy. We tested this proposal directly using macroautophagy-deficient fibroblasts. We found that rapamycin inhibits the aggregation of a fragment of huntingtin (exon 1) containing 97 polyQs similarly in macroautophagy-proficient (Atg5(+/+)) and macroautophagy-deficient (Atg5(-/-)) cells. These data demonstrate that autophagy is not the only mechanism by which rapamycin can alleviate the accumulation of misfolded proteins. Our data suggest that rapamycin inhibits mutant huntingtin fragment accumulation due to inhibition of protein synthesis. A model illustrates how a modest reduction in polyQ synthesis can lead to a long-lasting reduction in polyQ aggregation. We propose that several mechanisms exist by which rapamycin reduces the accumulation and potential toxicity of misfolded proteins in diseases caused by protein misfolding and aggregation.

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Autophagy, 4, 4, 542-5, 2008

PMID: 18418060

Open Access

The chemotactic behavior of computer-based surrogate bacteria.
D Bray, MD Levin, K Lipkow

Chemotaxis is the process by which organisms migrate toward nutrients and favorable environments and away from toxins and unfavorable environments. In many species of bacteria, this occurs when extracellular signals are detected by transmembrane receptors and relayed to flagellar motors, which control the cell's swimming behavior.

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Current biology : CB, 17, 1, 12-9, 2007

PMID: 17208180
DOI: 10.1016/j.cub.2006.11.027

Open Access

Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.
M Grati, ME Schneider, K Lipkow, EE Strehler, RJ Wenthold, B Kachar

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of approximately 5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 microm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that approximately 60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 microm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.

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The Journal of neuroscience : the official journal of the Society for Neuroscience, 26, 23, 6386-95, 2006

PMID: 16763047
DOI: 10.1523/JNEUROSCI.1215-06.2006

Open Access

Changing cellular location of CheZ predicted by molecular simulations.
Lipkow K

In the chemotaxis pathway of the bacterium Escherichia coli, signals are carried from a cluster of receptors to the flagellar motors by the diffusion of the protein CheY-phosphate (CheYp) through the cytoplasm. A second protein, CheZ, which promotes dephosphorylation of CheYp, partially colocalizes with receptors in the plasma membrane. CheZ is normally dimeric in solution but has been suggested to associate into highly active oligomers in the presence of CheYp. A model is presented here and supported by Brownian dynamics simulations, which accounts for these and other experimental data: A minority component of the receptor cluster (dimers of CheA(short)) nucleates CheZ oligomerization and CheZ molecules move from the cytoplasm to a bound state at the receptor cluster depending on the current level of cellular stimulation. The corresponding simulations suggest that dynamic CheZ localization will sharpen cellular responses to chemoeffectors, increase the range of detectable ligand concentrations, and make adaptation more precise and robust. The localization and activation of CheZ constitute a negative feedback loop that provides a second tier of adaptation to the system. Subtle adjustments of this kind are likely to be found in many other signaling pathways.

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PLoS computational biology, 2, 1553-7358, e39, 2006

PMID: 16683020

Open Access

Simulated diffusion of phosphorylated CheY through the cytoplasm of Escherichia coli.
K Lipkow, SS Andrews, D Bray

We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.

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Journal of bacteriology, 187, 1, 45-53, 2005

PMID: 15601687
DOI: 10.1128/JB.187.1.45-53.2005

Open Access

Early intermediates of mariner transposition: catalysis without synapsis of the transposon ends suggests a novel architecture of the synaptic complex.
K Lipkow, N Buisine, DJ Lampe, R Chalmers

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.

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Molecular and cellular biology, 24, 18, 8301-11, 2004

PMID: 15340089
DOI: 10.1128/MCB.24.18.8301-8311.2004

Open Access

Promiscuous target interactions in the mariner transposon Himar1.
K Lipkow, N Buisine, R Chalmers

We have previously characterized the early intermediates of mariner transposition. Here we characterize the target interactions that occur later in the reaction. We find that, in contrast to the early transposition intermediates, the strand transfer complex is extremely stable and difficult to disassemble. Transposase is tightly bound to the transposon ends constraining rotation of the DNA at the single strand gaps in the target site flanking the element on either side. We also find that although the cleavage step requires Mg2+ or Mn2+ as cofactor, the strand transfer step is also supported by Ca2+, suggesting that the structure of the active site changes between cleavage and insertion. Finally, we show that, in contrast to the bacterial cut and paste transposons, mariner target interactions are promiscuous and can take place either before or after cleavage of the flanking DNA. This is similar to the behavior of the V(D)J system, which is believed to be derived from an ancestral eukaryotic transposon. We discuss the implications of promiscuous target interactions for promoting local transposition and whether this is an adaptation to facilitate the invasion of a genome following horizontal transfer to a new host species.

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The Journal of biological chemistry, 279, 47, 48569-75, 2004

PMID: 15333635
DOI: 10.1074/jbc.M408759200

Open Access

Complete nucleotide sequence of Tn10.
R Chalmers, S Sewitz, K Lipkow, P Crellin

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

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Journal of bacteriology, 182, 10, 2970-2, 2000

PMID: 10781570

Open Access

Simulating the Bacterial Chemotaxis Pathway at High Spatio-temporal Detail
Sewitz S and Lipkow K

We have used the Smoldyn software to create spatially detailed models of the Escherichia coli chemotaxis pathway. With it we can follow signalling reactions at high spatio-temporal resolution, observe the formation of gradients of phospho-proteins as well as total protein and analyse the effects of macromolecular crowding on signalling. It has enabled us to propose new regulatory elements of the signalling pathway, which are mediated through the dynamic localisation and activity of the CheZ phosphatase. We used the Smoldyn software to model quantitative fluorescent microscopy data and to determine diffusion coefficients and binding affinities. We can reliably gain information obtained under conditions with high levels of experimental noise. Smoldyn can accommodate various cellular architectures, and we show that cell shape becomes an integral part of the signalling process.

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Current Chemical Biology, 7, 3, 214 - 223, 2013

PMID: