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A facilitated diffusion confusion dissolution

June 10, 2013 By Leave a Comment

To udirectbindfd1tilize the information contained within a cell’s genes, the enzyme RNA polymerase must find the beginning of each gene (the promoter).  Finding the beginning is a prodigious task:  RNAP must start at a particular base pair of DNA, but the cell contains millions of base pairs to choose from.  It has been proposed that gene-finding challenge is aided by a process termed ‘facilitated diffusion’ (FD).  In FD, RNA polymerase first binds to a random position on DNA and then slides along the DNA like a bead on a string until it encounters the target DNA sequence.

single-mol-testIn a recently published study in PNAS (1), biophysicists Larry Friedman and Jeffrey Mumm worked with Prof. Jeff Gelles in the Brandeis Biochemistry department to test key predictions of the FD model.  They used a novel light microscope that Friedman and colleagues invented and built at Brandeis, a microscope that can directly observe the binding of an individual RNA polymerase to a single DNA.  The scientists studied the σ54 RNA polymerase holoenzyme, an RNA polymerase found in most species of bacteria.  Surprisingly, none of the three predictions of the FD model that the experiments tested were found to be valid, demonstrating that target finding by the polymerase is not accelerated by sliding along DNA.  Friedman and colleagues instead propose that RNA polymerases are present in such large numbers that they can diffuse through the cell and efficiently bind to their target sites directly.  The absence of FD may explain how other proteins can bind to positions on the DNA that flank gene start sites and yet not interfere with RNA polymerase finding the gene.

Is this the end of the story? Not likely, given previous publications suggesting FD plays a role for some other DNA binding proteins. Using single-molecule techniques like those developed in the Gelles lab, scientists in next few years should give us a better idea if FD is very rare or very common. [editor: as a chemical engineer, I'm sad to see FD not have a role -- it seemed like such a nice theory...]

Friedman LJ, Mumm JP, Gelles J. RNA polymerase approaches its promoter without long-range sliding along DNA.  Proc Natl Acad Sci U S A. 2013 May 29. [Epub ahead of print]

 

 

Filed Under: Biochemistry, labs Tagged With: , , , , , , ,

Making new synapses with Sema4D

May 26, 2013 By

There are two main types of synaptic connections in the mammalian brain: excitatory glutamatergic synapses and inhibitory GABAergic synapses. The balance between excitatory and inhibitory inputs a neuron receives regulates the overall activity of neuronal networks; disruptions to this balance can cause epilepsy.

A new paper in J. Neuroscience from the Paradis lab shows that treatment of cultured neurons with the extracellular domain of the protein Sema4D causes a rapid increase (i.e. within 30 minutes) in the density of functional GABAergic synapses. Further, addition of Sema4D to neurons drives GABAergic synapse formation through a previously unappreciated mechanism: the splitting of pre-existing assemblies of the Gephyrin scaffolding protein. To our knowledge this is the fastest demonstration of synapse formation reported thus far and has significant implications for our understanding of the mechanisms of GABAergic synapse formation.

Screen Shot 2013-05-26 at 5.03.05 PMWhile the underlying mechanism of epileptogenesis is largely unknown, recurrent seizures emerge when there is an increase in network activity. One possible therapeutic treatment would be to restore normal network activity by increasing network inhibition. In an in vitro model of epilepsy, acute treatment with the protein Sema4D rapidly silences neuronal hyperexcitability, suggesting a possible use of Sema4D as a disease-modifying treatment for epilepsy.

Lead authors on the paper were Marissa Kuzirian, a grad student in the Neuroscience Ph.D. program, and Anna Moore, a Brandeis Neuroscience postdoctoral fellow.

Filed Under: Biology, grad students, Neuroscience, postdocs Tagged With: , , , , , , ,

More science

May 25, 2013 By

We’ve all been busy this spring writing grants and teaching courses and doing research and graduating(!), so lots of publications snuck by that we didn’t comment on. Here’s a few I think that might be interesting to our readers.

  • From Chris Miller‘s lab, bacterial antiporters do act as “virtual proton efflux pumps”:
  • nsrv2Are ninja stars responsible for controlling actin disassembly? Seems like the Goode lab might think so.
    • Chaudhry F, Breitsprecher D, Little K, Sharov G, Sokolova O, Goode BL. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin. Mol Biol Cell. 2013;24(1):31-41.
  • What do you get from statistical mechanics of self-propelled particles? The Hagan and Baskaran groups team up to find out.
  • From John Lisman and Ole Jensen (PhD ’98), thoughts about what the theta and gamma rhythms in the brain encode
  • From Mike Marr‘s lab, studeies using genome-wide nascent sequencing to understand how transcrption bursting is controlled in eukaryotic cells
  • From the Lau and Sengupta labs, RNAi pathways contribute to long term plasticity in worms that have gone through the Dauer stage
    • Hall SE, Chirn GW, Lau NC, Sengupta P. RNAi pathways contribute to developmental history-dependent phenotypic plasticity in C. elegans. RNA. 2013;19(3):306-19.
  • Can nanofibers selectively disrupt cancer cell types? Early results from Bing Xu‘s group.
    • Kuang Y, Xu B. Disruption of the Dynamics of Microtubules and Selective Inhibition of Glioblastoma Cells by Nanofibers of Small Hydrophobic Molecules. Angew Chem Int Ed Engl. 2013.

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Dogic Lab Wins Andor Insight Award

April 23, 2013 By

The ‘Insight Awards‘  is a video contest showcasing research imagery from the physical and life sciences which utilize Andor technology to capture data.  This year, the Dogic Lab submitted a research video to the competition and garnered first prize in the Physical Sciences division for their video of Oscillating Microtubule Bundles.

From the competition notes:

Microtubules are a bio-polymer composed of the protein tubulin and are used extensively in the cell for cellular division, cell motility, and transportation of cargo within the cell. Here, we investigate the material properties of mixtures of microtubules, a depletion agent, and the molecular motor Kinesin. The microtubules, driven by Kinesin motors, spontaneously organize into bundles of microtubules that oscillate in a manner reminiscent of flagella and cilia found in biology. This engineered system will allow us to studying systems of self-propelled and self-organized matter that exist far from equilibrium in the field known as Active Matter.

We use standard fluorescent microscopy to image labeled microtubules in a thin, flow cell microscope chamber. An Andor Clara camera was used in conjunction with a Nikon Ti Eclipse microscope to capture this video.

Video and Entry by Stephen DeCamp.

For this, and more videos from the Dogic Lab, visit their YouTube page or their website at Brandeis University.

Filed Under: awards, grad students, labs, Physics, Quantitative BIology Tagged With: , , , , , , , , , ,

Biogen Idec seeks regulatory approval for rFVIIIFc to treat Hemophilia A

March 13, 2013 By

Driving into work this morning, I heard that Biogen Idec is seeking FDA approval for a new breakthrough drug to treat Hemophilia A. The drug, a recombinant factor VIII Fc fusion protein (rFVIIIFc), provides a longer-lasting version of the clotting factor that is missing in these patients, and could help significantly reduce the frequency of injections for these patients (a big deal for children with an inherited bleeding disorder!)

The fusion protein approach is based on technology developed in part at Brandeis, by a collaboration involving Neil Simister‘s laboratory and researchers at Children’s Hospital and Brigham and Women’s Hospital, then further developed by a spinoff company, Syntonix Pharmaceuticals. Syntonix was acquired by Biogen Idec in 2007.

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Taste affects your sense of smell in the olfactory cortex

December 6, 2012 By

Professor Don Katz’s lab is interested in learning and behavior related to the gustatory system (the sense of taste). In a new paper in Journal of Neuroscience, also covered by the Washington Post website, Katz and  postdoc Joost Maier together with Univ. of Utah professor Matt Wachowiak, studied how tastes affect the processing of odors.

When any animal eats, it both smells and tastes the food, and has to make a split-second decision — is it nutritious or poisonous? Do I swallow it or spit it out? Accordingly, there has to be a processing system in the brain to integrate the information and make rapid decisions. It has been known for some time that odors affect the processing of taste in gustatory cortex. In the new article, the researchers demonstrate the effects of taste inputs on olfactory cortex. According to Maier, “this means is that the different senses are really interacting with each other at a much earlier level than previously thought,”.

Filed Under: labs, Neuroscience, Psychology Tagged With: , , , ,

Materials in Motion: Engineering Bio-Inspired Motile Matter

November 7, 2012 By

Life is on the move! Motion is ubiquitous in biology. From the gargantuan steps of an elephant to the tiniest single celled amoeba, movement in biology is a complex phenomenon that originates at the cellular level and involves the organization and regulation of thousands of proteins. These proteins do everything from mixing the cytoplasm to driving cell motility and cell division. Deciphering the origins of motion is no easy feat and scientists have been studying such complex behavior for quite some time. With biology as an inspiration, studying these complex behaviors provides insight into engineering principals which will allow researchers to develop an entirely new category of far-from-equilibrium materials that spontaneously move, flow or swim.

In a recent report in the journal Nature, a team of researchers from Brandeis University consisting of Tim Sanchez, Daniel T. N. Chen, Stephen J. DeCamp, Michael Heymann, and Zvonimir Dogic have constructed a minimal experimental system for studying far-from-equilibrium materials. This system demonstrates the assembly of a simple mixture of proteins that results in a hierarchy of phenomena. This hierarchy begins with extending bundles of bio-filaments, produces networks that mix themselves, and finally culminates in active liquid crystals that impart self-motility to large emulsion droplets.

Their system consists of three basic components: 1) microtubule filaments, 2) kinesin motor proteins which exert forces between microtubule filaments, and 3) a depletion agent which bundles microtubule filaments together. When put together under well-defined conditions, these components form bundled active networks (BANs) that exhibit large-scale spontaneous motion driven by internally generated active stresses. These motions, in turn, drive coherent fluid flows. These features bear a striking resemblance to a biological process called cytoplasmic streaming, in which the cellular cytoskeleton spontaneously mixes its content. Additionally, the system has great potential for testing active matter theories because the researchers can precisely tune the relevant system parameters, such as ATP and protein concentration.

 

The researchers also demonstrate the utility of this biologically-inspired synthetic system by studying materials science topics that have no direct biological analog. Under dense confinement to an oil-water interface, microtubule bundles undergo a spontaneous transition to an aligned state. Soft matter physics describes such materials as liquid crystals, which are the materials used to make liquid crystal displays (LCDs). These active liquid crystals show a rich variety of dynamical behavior that is totally inaccessible to their equilibrium analogs and opens an avenue for studying an entirely new class of materials with highly desirable properties.

Lastly, inspired by streaming flows that occur in cells, the researchers encapsulate the bundled active networks into spherical emulsion droplets. Within the droplet, microtubules again formed a self-organized nematic liquid crystal at the oil-water interface. When the droplets were partially squished between glass plates, the streaming flows generated by the dynamic liquid crystals lead to the emergence of spontaneous self-motility.

This research constitutes several important advances in the studies of the cytoskeleton, non-equilibrium statistical mechanics, soft-condensed matter, active matter, and the hydrodynamics of fluid mixing. The researchers have demonstrated the use of biological materials to produce biomimetic functions ranging from self-motility to spontaneous fluid flows using fundamentally new mechanisms. Additionally, the experimental system of bundled active microtubules is poised to be a model for exploring the physics of gels, liquid crystals, and emulsions under far-from-equilibrium conditions.

To see more videos from the Dogic lab at Brandeis University, check out their YouTube page.

Filed Under: Biochemistry, Biology, chemistry, labs, Physics, Quantitative BIology Tagged With: , , , , , , , ,

Easy Come, Easy Go

October 5, 2012 By

Whereas the diffusion of water molecules in the bulk liquid depends entirely on breaking hydrogen bonds, the diffusion of proton defects (i.e., an excess proton in acid or a proton deficit in base) is expedited by proton hopping across hydrogen bonds.  The details of this process are well understood in acid, and the process in base was believed to occur in analogous fashion. However, theoretical studies of hydroxide have given highly divergent predictions of solvation structures and diffusion rates, depending on the chosen recipe for such simulations: some predicted the traditionally expected solvation structures and some predicted the experimentally observed diffusion trends, but none do both. Now Seyit Kale, a graduate student in Prof. Judith Herzfeld’s group, has studied proton defects using the group’s recently developed LEWIS force field.[1] The LEWIS simulations obtain the correct relative diffusion rates with hydroxide solvation structures that are analogous to those of hydronium,[2] thereby supporting the traditional picture of the “proton hole”. The authors also catch and characterize proton transfer events, identifying similar “special pairs”[3] as the intermediates in both cases (see figure).

[1]       S. Kale, J. Herzfeld, J. Chem. Phys. 2012, 136, 084109.
[2]       S. Kale, J. Herzfeld, Angew. Chem. Int. Edit. 2012 in press. DOI: 10.1002/anie.201203568.
[3]       O. Markovitch, H. Chen, S. Izvekov, F. Paesani, G. A. Voth, N. Agmon, J. Phys. Chem. B. 2008, 112, 9456-9466.

 

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