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9 Publications

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    11/25/10 | Nanoscale architecture of integrin-based cell adhesions.
    Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM
    Nature. 2010 Nov 25;468(7323):580-4. doi: 10.1038/nature09621

    Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity, and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signaling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a \~{}40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signaling layer containing integrin cytoplasmic tails, focal adhesion kinase, and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

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    Gonen Lab
    11/25/10 | Tension directly stabilizes reconstituted kinetochore-microtubule attachments.
    Akiyoshi B, Sarangapani KK, Powers AF, Nelson CR, Reichow SL, Arellano-Santoyo H, Gonen T, Ranish JA, Asbury CL, Biggins S
    Nature. 2010 Nov 25;468(7323):576-9. doi: 10.1038/nature09594

    Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ’bi-oriented’ kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

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    Tjian Lab
    11/19/10 | A new focus on plant sciences.
    McCormick SJ, Tjian R
    Science. 2010 Nov 19;330(6007):1021. doi: 10.1073/pnas.1100640108
    Gonen Lab
    11/10/10 | Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.
    Wisedchaisri G, Dranow DM, Lie TJ, Bonanno JB, Patskovsky Y, Ozyurt SA, Sauder JM, Almo SC, Wasserman SR, Burley SK, Leigh JA, Gonen T
    Structure. 2010 Nov 10;18(11):1512-21. doi: 10.1016/j.str.2010.08.014

    Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins–the nitrogen regulatory protein NrpR–that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.

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    Looger Lab
    11/10/10 | Toward the second generation of optogenetic tools.
    Knöpfel T, Lin MZ, Levskaya A, Tian L, Lin JY, Boyden ES
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2010 Nov 10;30(45):14998-5004. doi: 10.1523/JNEUROSCI.4190-10.2010

    This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.

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    11/01/10 | Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography.
    Amat F, Comolli LR, Nomellini JF, Moussavi F, Downing KH, Smit J, Horowitz M
    Journal of Bacteriology. 2010 Nov;192(22):5855-65. doi: 10.1128/JB.00747-10

    The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.

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    Looger LabSvoboda Lab
    11/01/10 | Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.
    Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW
    Nature Neuroscience. 2010 Nov;13(11):1433-40. doi: 10.1038/nn.2648

    Spatial navigation is often used as a behavioral task in studies of the neuronal circuits that underlie cognition, learning and memory in rodents. The combination of in vivo microscopy with genetically encoded indicators has provided an important new tool for studying neuronal circuits, but has been technically difficult to apply during navigation. Here we describe methods for imaging the activity of neurons in the CA1 region of the hippocampus with subcellular resolution in behaving mice. Neurons that expressed the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-restrained mice performed spatial behaviors in a setup combining a virtual reality system and a custom-built two-photon microscope. We optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit. The combination of virtual reality and high-resolution functional imaging should allow a new generation of studies to investigate neuronal circuit dynamics during behavior.

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    11/01/10 | Imaging: visualizing the possibilities.
    Lippincott-Schwartz J
    Journal of cell science. 2010 Nov 1;123(Pt 21):3619-20. doi: 10.1242/jcs081539
    Svoboda Lab
    11/01/10 | The functional asymmetry of auditory cortex is reflected in the organization of local cortical circuits.
    Oviedo HV, Bureau I, Svoboda K, Zador AM
    Nature Neuroscience. 2010 Nov;13(11):1413-20. doi: 10.1038/nn.2659

    The primary auditory cortex (A1) is organized tonotopically, with neurons sensitive to high and low frequencies arranged in a rostro-caudal gradient. We used laser scanning photostimulation in acute slices to study the organization of local excitatory connections onto layers 2 and 3 (L2/3) of the mouse A1. Consistent with the organization of other cortical regions, synaptic inputs along the isofrequency axis (orthogonal to the tonotopic axis) arose predominantly within a column. By contrast, we found that local connections along the tonotopic axis differed from those along the isofrequency axis: some input pathways to L3 (but not L2) arose predominantly out-of-column. In vivo cell-attached recordings revealed differences between the sound-responsiveness of neurons in L2 and L3. Our results are consistent with the hypothesis that auditory cortical microcircuitry is specialized to the one-dimensional representation of frequency in the auditory cortex.

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