Collaboration & Service Projects

MMBioS will engage in multiple, diverse Collaboration and Service projects (C&SP).  These C&SPs  will benefit from the tools and technologies developed by MMBioS.  Current CS&Ps are listed below; this list will grow over time.

 


 

1) Relationship between structural Dynamics and Sequence Evolution; Application to molecular machined such as bacterial chaperonons and HSP70 chaperones

PI: I. Bahar, University of Pittsburgh  Collaborators: Amnon Horovitz, Weizmann Institute; R. Altman, Stanford University; L. Gierasch, University of Massachusetts.

aanm We have expanded the scope of this project to include Dr. Amnon Horovitz from Weizmann Institute, Israel, an expert in the allostery of chaperonins as well as sequence co-evolution. We are exploring the sequence->structure->dynamics->function mapping of allosteric proteins. Our overarching goal is to establish the computational methodology for simulating the machinery of biomolecular systems on the order of Megadaltons. The molecular machines that we selected for developing and implementing the methodology are bacterial chaperonins (GroEL), molecular chaperones (E. coli Hsp70 DnaK) and their co-chaperones. We study the ATP-regulated mechanisms of intersubunit (GroEL) or interdomain (DnaK) communication, as well as the role of co-chaperonins or co-chaperones in modulating the conformational changes occurring along the allosteric cycle of these machines.

In Year 3 we made progress in quantitative analysis of the evolution of proteins, and in particular in assessing the relationship, if any, between their structural dynamics and sequence evolution. In a recent study performed toward this goal (Mao et al., 2015), we carried out a systematic comparative analysis of current methods for analyzing sequence co-evolution and we proposed a hybrid model that yields high performance. The two metrics for performance were the elimination of false positives between non-interacting proteins, and the verification of tertiary contacts between co-evolving pairs. The methods used in this newly published (that acknowledged MMBioS support) are currently being implemented into our ProDy API.

Publications Resulting from This Work

    • General IJ, Liu Y, Blackburn M, Mao W, Gierasch LM, Bahar I (2014) ATPase subdomain IA is a mediator of interdomain allostery in Hsp70 molecular chaperones PLoS Comp Bio 10: e1003624 PMID: 24831085, PMC4022485
    • Mao W, Kaya C, Dutta A, Horovitz A, Bahar I (2015) Comparative Study of the Effectiveness and Limitations of Current Methods for Detecting Sequence Coevolution Bioinformatics 31: 1929-37 PMID: 25697822; PMC4481699

Completed


 

2) Membrane Proteins Structure and Dynamics Consortium (MPSDC) Computational Core, and AMPA receptors structural dynamics

PI: I. Bahar, University of Pittsburgh   Collaborators: B. Roux and E. Perozo, University of Chicago; K. Schulten and E. Tajkhorshid, University of Illinois at Urbana-Champaign;  I. Greger, University of Cambridge, UK; H. Weinstein, Cornell University

To assess whether ANM-predicted AMPAR NTD pivoting motions can occur in cellulo, the Greger lab mutated K262 to cysteine (K262C), hypothesizing that this mutation would trap the NTD tetramer in a closed conformer via formation of a disulfide bridge. GluA2 wild type (WT) and K262C mutant were expressed in HEK293 cells and protein extracts were analyzed by Western blot. The results showed that our hypothesis was valid, thus lending support to computational predictions. We further examined the dynamics of AMPAR and NMDAR to observe that the differ along a few slow modes only (Figure 1). Overall, our data provided a first glimpse into the dynamic spectrum of AMPAR and NMDARs and delineated conserved mechanisms underlying allosteric communication in iGluRs.

csp2Figure 1. AMPAR to NMDAR conformations differ by rearrangements along a few soft modes. The dark blue bars show the overlap (correlation cosine) between ANM modes of AMPAR and the structural difference vector between AMPAR and NMDAR; the red curve displays the cumulative overlap; the dotted green curve shows the expected cumulative overlap if the modes were equally contributing each. The inset shows the initial structures of AMPAR (left) and NMDAR (right).

Publications Resulting from This Work

    • Krieger J, Bahar I, Greger IH (2015) Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains Biophys J109: 1136-48 PMID: 26255587, PMC4576161
    • Dutta A, Krieger J, Lee JY, Garcia-Nafria J, Greger IH, Bahar I (2015) Cooperative Dynamics of Intact AMPA and NMDA Glutamate Receptors: Similarities and Subfamily-Specific Differences Structure23: 1692-1704 PMID: 26256538, PMC4558295
    • Dutta A, Shrivastava IH, Sukumaran M, Greger IH, Bahar I (2012) Comparative dynamics of NMDA- and AMPA-Glutamate receptor N-terminal domains Structure20: 1838-49 PMID: 22959625, PMC3496038

Completed

 


 

3) Integration, prediction, and generation of mixed mode information using graphical models, with applications to protein-protein interactions

PIs: C. J. Langmead, Carnegie Mellon University Collaborators: C. Bailey-Kellog, Dartmouth University; N. Ramakrishnan, Virginia Tech;  A. Friedman, Purdue University

This C&SP was an NSF-funded project titled: “Integration, prediction, and generation of mixed mode information using graphical models, with applications to protein-protein interactions” (NSF IIS-0905193). It was collaboration between Carnegie Mellon University, Dartmouth College, Virginia Tech, and Purdue. The project concluded in August, 2013. The focus of this C&SP was to develop novel probabilistic graphical models for modeling protein-protein interactions. Three broad classes of techniques were developed to integrate attribute-value and relational information, integrate statistical and physical information, and utilize probabilistic models generatively. The research has resulted in new graphical models, new algorithms, and the application of those algorithms to the modeling of protein structures and dynamics, including protein-protein interactions. Specific accomplishments include: the first optimal algorithm for learning regularized undirected graphical models of protein sequences; the first method for performing binding free energy calculations for protein-protein interactions via graphical models; the first probabilistic graphical models of molecular dynamics; the first algorithm for learning the parameters of molecular mechanics force fields by minimizing functionals over Boltzmann distributions; the first undirected model of distributions on the hypersphere (for modeling distributions over angles); a game-theoretic method for modeling the emergence of drug resistance-causing mutations in proteins; new algorithms for learning semi- and non-parametric distributions; and a new class of regression models for predicting binding free energies. The project resulted in seventeen publications, three Ph.D. dissertations, and one patent. Primary applications of this research include computer-aided drug design and computer-aided protein engineering. Secondary applications of this work include techniques for identifying functionally important residues mediating binding and allostery.

Publications Resulting from This Work

    • Kamisetty H, Ghosh B, Langmead CJ, Bailey-Kellogg C (2014) Learning Sequence Determinants of Protein: protein Interaction Specificity with Sparse Graphical Models Res Comput Mol Biol8394:129-143 PMID: 25414914, PMC4235964.
    • Kamisetty H, Ghosh B, Langmead CJ, Bailey-Kellogg C (2015) Learning sequence determinants of protein: protein interaction specificity with sparse graphical models J Comput Biol22:474- 86 PMID: 25973864, PMC4449715

Completed


 

4) Calcium entry and transmitter release at the neuromuscular junction

PIs: T. Bartol, T. Sejnowski, Salk Institute; R. Laghaei, G. Hood, M. Dittrich, Pittsburgh Supercomputing Center  Collaborator: S. Meriney, University of Pittsburgh

Communication between cells in the nervous system (synaptic transmission) underlies all complex behaviors, is often disrupted in neurological disease, and is a focus for therapeutic intervention. Synapses work by releasing chemical transmitter from a region called the active zone. In this project we synergistically combine computer simulation using MCell (Dittrich lab) and synaptic anatomy, physiology, and Ca2+ imaging (Meriney lab) to investigate the structure and function of synapses. We have previously completed and published a baseline model of frog neuromuscular junction (NMJ) active zone structure and function (Dittrich et al., 2013). Based on this model we have recently investigated several possible mechanisms underlying short-term synaptic facilitation at the frog NMJ (Ma et al., 2015). Our study showed that a vesicle release mechanism in which a second set of Ca2+ sensor sites was responsible for facilitation provided good overall agreement with our experimental constraints. In addition, we used our MCell modeling approach combined with Ca2+ imaging, pharmacological Ca2+ channel block, and postsynaptic recording to show that release of individual synaptic vesicles at the frog NMJ is predominately triggered by Ca2+ ions entering the nerve terminal through the nearest open calcium channel. This work has recently been published (Luo et al., 2015). Building on these insights, we are currently expanding our investigation to the role of detailed active zone structure in synapse function with a focus on neuromuscular diseases such as Lambert-Eaton Myasthenic Syndrome.

Publications Resulting from This Work

    • Tarr TB, Dittrich M, Meriney SD (2013) Are unreliable release mechanisms conserved from NMJ to CNS? Trends Neurosci36(1):14-22. PMID: 23102681, PMC4076818
    • Meriney SD, Dittrich M (2013) Organization and function of transmitter release sites at the neuromuscular junction J Physiol591(13):3159-65. PMID: 23613535, PMC3717219
    • Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD (2013) An excess-calcium-binding-site model predicts neurotransmitter release at the neuromuscular junction Biophys J104(12):2751-63 PMID: 23790384, PMC3686347
    • Ma J, Kelly L, Ingram J, Price TJ, Meriney SD, Dittrich M (2015) New insights into short-term synaptic facilitation at the frog neuromuscular junction J Neurophysiol113:71-87 PMID: 25210157, PMC4294575
    • Luo F, Dittrich M, Cho S, Stiles JR, Meriney SD (2015) Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction J Neurophysiol113:2480-9 PMID: 25652927, PMC4416571

Completed


 

5) Modeling immunoreceptor signaling, autophagy, and endocytosis, Hardening Software for Rule-Based Modeling

PI:  J. Faeder, University of Pittsburgh  Collaborator: W. S. Hlavacek, Los Alamos National Laboratory

The fundamental events in FcεRI signaling initiation were considered during the pioneering work of Goldstein, Faeder, Hlavacek and colleagues to establish the utility of rule-based methods for mechanistic modeling of the dynamics of molecular interactions in signaling networks. This led to the creation of BioNetGen, already introduced in the BTRC proposal as a computational framework for modeling biochemical networks. Here, we propose to take advantage of successful initiatives at the BTRC for integration of the BioNetGen rule-based modeling approach into the mesh-based architecture that underlies MCell spatial simulations. In particular, we will focus on the proposed new capabilities for adaptive meshing, which can superimpose well-characterized changes in surface geometry over time during simulations. This unique feature will allow us to develop novel computational models to describe simultaneously slow endocytic vesicle dyanamics and fast signaling dynamics at vesicle surfaces.

Aim 1. To utilize the CellBlender interface to create a framework for cell shape changes that occur during mast cell signaling, such as microvillar-to-ruffled transformation, appearance of signaling patches, and changes associated with enhanced endocytosis.

Aim 2. To evaluate the effects of a changing 3D geometry on signaling output, by incorporating existing Rule-Based Models for FcεRI chemical reaction networks into MCell-BioNetGen and refining the model with new parameters for spatial distributions, rate constants and diffusion characteristics collected through funded initiatives.

During the project we developed MCell-R, which now enables network-free simulations to be performed directly in MCell based on a BioNetGen model specification. We have validated that the simulator gives correct results for a range of FceRI signaling models and also for the bivalent-ligand bivalent receptor, a simple example of unrestricted aggregation. In principle the simulator can now be used to simulate spatial signaling dynamics of any model that can be described in BNGL with any geometry that can be specified in MCell, including a geometry that changes in time. We plan to implement several such models in the future.

Publications Resulting from This Work

    • Chylek LA, Harris LA, Tung CS, Faeder JR, Lopez CF, Hlavacek WS (2013) Rule-based modeling: a computational approach for studying biomolecular site dynamics in cell signaling systems Wiley Interdiscip Rev Syst Biol Med6: 13-36 PMID: 24123887, PMC3947470
    • Chylek LA, Harris LA, Faeder JR, Hlavacek WS (2015) Modeling for (physical) biologists: an introduction to the rule-based approach Phys Biol12: 045007 PMID: 26178138, PMC4526164

In progress


 

6) Distance-dependent structure and function of neuronal dendrites

PIs: T. Sejnowski and T. Bartol, The Salk Institute Collaborator: K. Harris, University of Texas at Austin

The purpose of this CS&P is to collaborate with Kristen Harris' lab to create realistic MCell models of dendritic structure and function.

MCell can simulate the diffusion and interaction of molecules involved in biochemical signaling pathways within the 3D subcellular structure of cells. To do so, surface meshes used to represent cell membranes and subcellular structures must meet very strict geometric standards (e.g. water-tight, non-intersecting, manifold). We help create realistic MCell models of CA1 dendritic spines, in particular to learn how more the accurate serial section tomographic reconstructions of core organelles impact simulations of molecular signaling within dendritic spines.

Recently two papers were published relating to this C&SP. The first paper, published in Frontiers in Synaptic Neuroscience, reported on reconstitution of calcium dynamics in dendritic spines. Nine parameters of the model were optimized within realistic experimental limits by a process that compared results of simulations to published data. Simulations in the optimized model reproduce the timing and amplitude of Ca(2+) transients measured experimentally in intact neurons. Thus, the characteristics of individual isolated proteins determined in vitro could accurately reproduce the dynamics of experimentally measured Ca(2+) transients in spines. The second paper, published in eLife, found that dendritic spines that receive input from the same axon are the same size. This finding allowed estimation of the variability of synaptic plasticity and we found that the amount of information stored at synapses is approximately 4.7 bits, which is an order of magnitude larger than previous estimates.

Publications Resulting from This Work

    • Edwards J, Daniel E, Kinney J, Bartol T, Sejnowski T, Johnston D, Harris K, Bajaj C (2014) VolRoverN: enhancing surface and volumetric reconstruction for realistic dynamical simulation of cellular and subcellular function Neuroinformatics12:277-89 PMID: 24100964, PMC4033674
    • Kinney JP, Spacek J, Bartol TM, Bajaj CL, Harris KM, Sejnowski TJ (2013) Extracellular sheets and tunnels modulate glutamate diffusion in hippocampal neuopil J Comp Neurol521: 448-64 PMID: 22740128, PMC3540825
    • Bartol TM, Keller DX, Kinney JP, Bajaj CL, Harris KM, Sejnowski TJ, Kennedy MB (2015) Computational reconstitution of spine calcium transients from individual proteins Front Synaptic Neurosci7: 17 PMID: 26500546, PMC4595661
    • Bartol TM, Bromer C, Kinney J, Chirillo MA, Bourne JN, Harris KM, Sejnowski TJ (2015) Nanoconnectomic upper bound on the variability of synaptic plasticity Elife4.pii: e10778 PMID: 26618907, PMC4737657

Completed


 

7) Interfacing image-derived generative models with cell simulation engines

PIs: R. Murphy and G. Rohde, Carnegie Mellon University; M. Dittrich, Pittsburgh Supercomputing Center; J. Faeder, University of PittsburghCollaborators: T. Bartol, The Salk Institute; S. Andrews and R. Brent, Fred Hutchison Cancer Research Center; I. Moraru, J. Schaff and L. Loew, University of Connecticut

The primary goal of this collaborative project is to develop methods to exchange generative models of the spatiotemporal organization of cells between various cell simulation engines. This will enable those engines to have a greatly expanded source of cell geometries, to take full advantage of dynamic spatial models, and to combine models constructed by various means. The vision is for CellOrganizer to be able to work synergistically with Virtual Cell, Smoldyn, MCell, and any other cell simulator. A secondary, but important, goal will be to establish a de facto standard by which future systems for constructing generative cell models can interface with simulation engines. The interfacing would go well beyond the SBML spatial package, consisting of modules to generate various types of models from different image sources, to save them in exchangeable files, and most importantly, plugins for simulators to generate instances from the models to use in simulations and specify how those instances change over time.

In progress


8) Using generative models of cell organization to investigate tumor cell heterogeneity

PIs: R. Murphy and G. Rohde, Carnegie Mellon University Collaborators: S. Altschuler and L. Wu, University of Texas Southwestern Medical Center

Tumor cell populations have long been known to be complex mixtures of multiple phenotypes. Given the precision with which one is able to measure properties of cellular structure and function, it is often the case that the measure of two cells will differ. Paramount to deriving useful information from a potentially large set of measurements is to be able to determine which differences are statistically significant as well as their biological meaning. The established framework for analyzing the heterogeneity of tumor cell populations involves analyzing these measurements in a high dimensional feature space. Features can be simple and easily interpretable (such as cell size), or complex and difficult to interpret (such as texture features). The goal of this subproject is to try to represent cell images using generative models that are more easily interpretable and could potentially offer more insights into the underlying phenomena that affect the distribution of phenotypes in tumor cells.

In progress


 

9) Large-scale electron microscopy of calcium-imaged neuron populations

PIs: A. Wetzel and G. Hood, Pittsburgh Supercomputing Center Collaborator: D. Bock, Janelia Farm Research Campus, Howard Hughes Medical Institute

During the last year of this project, the biological focus of Bock's work has shifted from localized mouse brain circuits to the mapping of complete drosophila brains with an emphasis on visual circuits. This change did not affect the relation of our collaboration which is focused on the technical aspects of maximum scale serial-TEM data assembly.

Bock's group has improved the throughput of their automated 4 camera TEM system. Although the cameras and their raw data rates are unchanged new mechanisms have been tested in the last year for automatic sample cassette loading. This has substantially improved the ratio of imaging to sample exchange time. The long term goal for this new sample loading method is to enable continuous runs up to 2 weeks for datasets of 15,000 sections and >50 TBytes. Individual sections are imaged in arrays of ~20,000 5 MPixel tiles, typically 112 columns by 196 rows, with a unique stepping pattern to accommodate constraints on the spacing of the optical cameras and lenses. One difficult aspect of these data is the use of the small 5 MPixel tiles which, particularly in the case of hierarchical registration methods, become limited by poor performance of large filesystems when processing small files.

Regular production of datasets at this scale would be difficult to register using our previous method of raw data transmission to the PSC followed by the return of aligned results to Janelia Farm. Therefore we are preparing for Bock's team to run our alignment codes on the large 6,144 core Janelia Farm computing cluster. Wetzel and Hood were part of an April 1-11, 2015 Janelia Farm image registration Hackathon organized by Stephan Saafeld's group. During that trip we were able to test both the AlignTK and SWiFT codes on the Janelia systems using data from Bock's work as well as many others from Janelia and elsewhere (i.e. Winfried Denk and our C&SP10 collaborator Jeff Lichtman). We were also able to adapt a key component of the SWiFT method for use within one of the Janelia registration codes and found that it greatly improved the reliability of registration on difficult regions of Bock's data.

Publications Resulting from This Work

    • Bock DD, Lee WC, Kerlin AM, Andermann ML, Hood G, Wetzel AW, Yurgenson S, Soucy ER, Kim HS, Reid RC (2011) Network anatomy in vivo physiology of visual cortical neurons Nature471: 177-82 PMID: 21390124, PMC3095821

Completed


 

10) Advancing high-throughput thin-section scanning EM to study relationships between neuronal circuit structure and function

PIs: A. Wetzel and G. Hood, Pittsburgh Supercomputing Center Collaborator: J. Lichtman, Harvard University

We have continued work on automated alignment of serial SEM image sets produced using tape collecting ultramicrotome sectioning and wafer mounted imaging procedures developed at Harvard. The primary datasets have been the 16,000 section zebrafish stack, which is the focus of a new CS&P project with the Engert lab and the 114 TB lateral geniculate nucleus (LGN) volume that was previously acquired and is the focus of this C&SP.

The different requirements and resolutions of these datasets have highlighted the need for a variety of approaches to handle different types of biological content and different types of image characteristics. The large number of tape cracks in the LGN dataset has been particularly difficult since the cracks from any section to its neighbors interfere with one another during the alignment process. Even though the detailed cracking pattern is different on each section the fact that most of the cracks have similar sizes and orientations means that they tend to pull the alignment such that we have seen errors on the order of 1 per 1000 sections. We are currently working on a strategy to produce a low-resolution map of the cracks, 1/4th scale, that can then be used to constrain the SWiFT alignment process.

We are entering a new and much higher-throughput phase of EM data capture. Previous datasets, including the zebrafish and LGN, were captured over several months using a Zeiss Merlin microscope and pixel rates up to 20MHz. Lichtman's team recently installed the first Zeiss 61-beam 1.2 Gpixel/sec scanning microscope. This microscope is now being tested with large single sections in the 400GB/section range, which are captured in a hexagonal mosaic pattern. These data will require a substantial number of registration code adaptations. We will test speed and accuracy of these modifications once the new equipment is producing serial spans of at least 100 sections, which is where the power of the SWiFT approach is most useful.

Publications Resulting from This Work

    • Morgan JL, Berger DR, Wetzel AW, Lichtman JW (2016) The fuzzy logic of network connectivity in mouse visual thalamus Cell165: 192-206 PMID: 27015312, PMC4808248
    • Hildebrand DGC, Torres RM, Choi W, Tran Minh Quan, Arthur Willis Wetzel, George ScottPlummer, Ruben Portugues, Isaac Henry Bianco, Owen Randlett, Stephan Saalfeld, Alex Baden, Kunal Lillaney, Randal Burns,Joshua Tzvi Vogelstein, Won-Ki Jeong, Jeff William Lichtman, Florian Engert (2016) Whole-brain serial-section electron microscopy in larval zebrafish Nature, revised version in preparation.

Completed


 

11) Morphological and regulatory models of neuronal differentiation

PIs: H Busch and M Böerries, University of Freiburg Collaborators: R Murphy and G Rohde, Carnegie Mellon University

The goal of this project is to develop a spatiotemporal generative model of the changes in cell size, shape and subcellular organization that occur during the differentiation of PC12 cells induced by nerve growth factor. This model will be related to a regulatory model constructed from parallel measurements of RNA expression over the time course of differentiation. The results are expected to lead to the identification of gene expression changes that lead to specific morphological changes and to test their importance for the differentiation process.

Publications Resulting from This Work

    • Johnson GR, Bierschenk I, Nitschke R, Boerries M, Busch H, and Murphy RF. (2017) Image-derived Models of Cell Organization Changes During Differentiation of PC12 Cells, submitted.

Completed


12) Generative models of plant organelle distribution and differentiation

PI: K Palme, University of Freiburg Collaborator: R Murphy, Carnegie Mellon University

Plants exhibit the ability to undergo dramatic dedifferentiation and redifferentiation such that both protoplasts (cells extracted from mature plants) and microspores (cells that arise during gametogenesis) can give rise to mature plants under the right circumstances. The goal of this project is to identify the specific changes in protein expression and localization that are associated with such processes, in part by building spatiotemporal generative models directly from images and movies of cells obtained by light microscopy.

Publications Resulting from This Work

    • Johnson GR, Kangas JD, Dovzhekno A, Trojok R, Voigt K, Majarian TD, Palme K and Murphy RF. (2017) A Method for Characterizing Phenotypic Changes in Highly Variable Cell Populations and its Application to High Content Screening of Arabidopsis thaliana Protoplasts. Cytometry Part A, in press.

Completed


13) Microtubule pattern analysis and drug sensitivity

PI: P Giannakakou, Cornell University Collaborator: R Murphy, Carnegie Mellon University

The goal of this new project is to determine whether and how the distributions of microtubules differ between tumor cells sensitive and resistant to drugs that act on microtubules. Initial work has focused on training systems to recognize these patterns, with a complicating factor being the large differences in cell size, shape and unperturbed microtubule pattern between cells from different tumors. Preliminary results indicate that three broad but distinct patterns can be distinguished, corresponding to untreated cells, drug-treated resistant cells, and drug-treated sensitive cells. Future work will focus on using generative models to remove variation due to cell size and shape. Ultimately, this would be used to determine whether a particular tumor is likely to be resistant to a particular drug and thereby to choose an appropriate drug for that tumor.

Completed


 

14) Actin filament patterns and cell motility

PI: J Theriot, Stanford University Collaborators: G Rohde and D Slepčev, Carnegie Mellon University

This is a new project, the goal of which is to quantitatively describe the patterns of actin filaments and related proteins as they relate to cell motility in neutrophils. The project aims to use recently developed methods for measuring the similarity between such patterns, as well as transport-based method for finding correlation between intensity patterns, to decode relationships between different proteins as cells move. Results are expected is to help characterize cell motion as a function of the subcellular protein patterns.

Completed


15) Reconstructing zebrafish neural circuits controlling visually induced behaviors

PI: Art Wetzel and Greg Hood, Pittsburgh Supercomputing Center Collaborators: Florian Engert and David Hildebrand, Harvard University

The general goal of this new C&SP is the development of the larval zebrafish as a model system for the comprehensive identification and examination of neural circuits controlling visually induced behaviors. To that end our collaborators plan to establish and quantify a series of visually induced behaviours and analyze the individual resulting motor components. Using these assays they will monitor neuronal activity throughout the fish brain in an awake and intact preparation.

The underlying data are highly accurate zebrafish reconstructions from serial section SEM. The primary data is an 18,000 section series prepared by our collaborator David Hildebrand of a 6 day post fertilization specimen with full body cross section coverage extending from the front of the animal through the entire brain and into the initial spinal cord region. This SEM data consists of 3 resolutions 1) a very low resolution overview, ~1 micron/pixel, of the full18,000 section series that was used for ROI selection and targeting 2) a moderate resolution 60nm per pixel scan of full body cross sections over a 16,000 section ROI used for tracing myelinated pathways and 3) a 20 nm per pixel scan of a 12,000 section sub-region covering the entire brain that is used for detailed circuit tracing. As part of this collaboration we have applied our SWIFT approach to help produce highly accurate alignments of these image stacks. This required a number of significant improvements to the underlying core algorithms in our SWIFT software. Using SWIFT, we were already able to produce a very well aligned stack of the 60 nm image data shown in Figure 1 as a greatly reduced cut plane view. We aren currently using the SWIFT approach align the higher resolution 20nm stack, which is needed for accurate segmentation of the zebrafish brain area, into a geometry that is consistent with the lower resolution volumes.

The longer-term goal of correlating in-vivo neural activity with brain structure requires multi-modal alignment of EM data with 2-photon imaging as the animal reacts to visual stimuli. A preliminary example of the 2-photon data, acquired from a different specimen, is shown in Figure 2 with the primary forebrain ROI for in-vivo study indicated by the green square. After detailed reconstruction of pathways in the current dataset we will prepare a series of dual SEM plus 2-photo datasets for this forebrain region that will require accurate and efficient registration of the EM data for structural tracing along with alignment of the 2-photon data onto the EM data for correlation to in-vivo data recordings.

Publications Resulting from This Work

      • Hildebrand DGC, Torres RM, Choi W, Tran Minh Quan, Arthur Willis Wetzel, George ScottPlummer, Ruben Portugues, Isaac Henry Bianco, Owen Randlett, Stephan Saalfeld, Alex Baden, Kunal Lillaney, Randal Burns,Joshua Tzvi Vogelstein, Won-Ki Jeong, Jeff William Lichtman, Florian Engert (2016) Whole-brain serial-section electron microscopy in larval zebrafish Nature, revised version in preparation.

In progress


 

16) Latent Factor Models for Identification of Novel Neuroprotectives for Huntington's Disease

PI: Ivet Bahar and Bing Liu, University of Pittsburgh;  Collaborators: Robert Friedlander, Mark Shurdak, Andrew Stern, D. Lancing Taylor, University of Pittsburgh

This C&SP is supported by NIH award 5R01NS077748-04 (PI: Friedlander) entitled “Functional role of microRNAs in Huntington's disease pathogenesis”. Our focus is the identification of novel neuroprotectives using latent factor modeling (LFM). Firstly, we built a set of known neuroprotectives based on the data in the literature by collecting compounds that were identified in a large scale screen for neuroprotectives in Huntington’s disease (HD) as well as compounds that have been tested in clinical trials for their neuroprotective effect. Then, the LFM-based algorithm learns the most successful latent factor model of the drug-target interactions in the STITCH dataset after assessing the performance of multiple LFM learning algorithms. The LFM of STITCH allowed us to quantitatively assess the interaction profiles of the known neuroprotectives, which we then used to find new compounds toward (i) the discovery of new neuroprotectives by target-based diversification of known neuroprotectives, (ii) the discovery of mechanism of action hypotheses by identifying targets of interest. Specifically, for each target of known neuroprotectives, we identified other chemicals known to be interacting with high confidence and sorted them for maximal dissimilarity to the known neuroprotectives interacting with that target. This method identifies compounds that share the target of interest, but are otherwise as diverse in their interaction profile as possible. Compounds selected will be active if the target of interest is important for the HD etiology, therefore they represent new potential treatment candidates. Furthermore if one or more targets yield many neuroprotectives, that target is implicated as being central to the disease pathophysiology; therefore this method allows us to infer about the proteins regulating the disease phenotype by testing compounds alone.

Publications Resulting from This Work

      • Stern, A. M.; Schurdak, M. E.; Bahar, I.; Berg, J. M.; Taylor, D. L.(2016) A Perspective on Implementing a Quantitative Systems Pharmacology Platform for Drug Discovery and the Advancement of Personalized Medicine. J Biomol. Screen., in press. PMID: 26962875

In progress


 

17) Structure and Function of Voltage-Gated Calcium Channels

PI: Ivet Bahar and Mary Cheng, University of Pittsburgh; Tom Bartol, Salk Institute; Collaborators: Peter Wipf and Steve Meriney, University of Pittsburgh

This C&SP is a joint Charles E. Kaufman Foundation Grant proposal titled “Structure and Function of Voltage-Gated Calcium Channels (VGCCs)”, submitted on June 30 2015. The goal is to establish a chemistry - computational biology - neuroscience collaboration and launch a novel combination of strategic chemical synthesis (Wipf), VGCC mutagenesis and biophysical measurements of VGCC function (Meriney), and computational modeling (Bahar) toward the development a model of Cav2 VGCC gating. This work will fill a critical gap in our understanding of VGCC structure and function and significantly impact future studies in the field. VGCC are critical regulators of cellular function. We are interested in an important family of VGCCs (Cav2) that regulate communication between neurons in the brain. Despite its critical importance in cellular function, surprisingly little is known about VGCC structure and function, primarily because a crystal structure has proven very difficult to generate for this large membrane protein. In preliminary studies, the Wipf and Meriney labs have developed new tools targeted selectively to voltage-gated calcium channels. With the expertise of a new member of our team, Dr. Bahar, we propose to deploy novel chemical synthesis, calcium channel mutagenesis, biophysical measurements of calcium channel function, and computational modeling of channel movement to significantly increase our understanding of the structure-function relationships of voltage-gated calcium channels. This work will be transformative since it will impact the study of communication between cells, especially in the brain, and likely lead to the development of novel drugs that may be useful for a variety of diseases. Bahar lab will focus on structural modeling and simulations of VGCC gating and drug binding: a) modeling the structures and dynamics of the Cav2 VGCC in different conformational states, with a focus on key residues and interactions that control gating; and b) determination of the mechanism of action of the agonist gating modifiers of Cav2 VGCCs .

In progress


 

18) Effect of RNA-editing of ADAR1 on the activity of ionotropic glutamate receptors

PI: Ivet Bahar, Mary H Cheng, University of Pittsburgh  Collaborators: QingDe Wang, University of Pittsburgh

This is a C&SP, supported by NIH 5R21CA158650-02 (PI: Wang). The goal of the project is to define the function of RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) in hematopoietic and leukemia stem cells. ADAR1 is an essential protein for embryonic and adult hematopoiesis, while the leukemia cells are more susceptible to the gene deletion that codes ADAR1. The impact of ADAR1 on normal and leukemia stem cells (LSC) remain unknown, and there is a need to develop a therapeutic strategy to eliminate LSCs by targeting ADAR1. Although ADAR1 is identified as an RNA editing enzyme and RNA editing has been shown to play critical roles in stem cells and other biological processes, multiple attempts to date have been unable to identify an editing target that accounts for the death of normal hematopoietic and leukemia cells of ADAR1 knockouts. The R21 seeks a definitive answer whether the editing activity of ADAR1 is necessary for the proliferation and differentiation of LSCs.

ADAR has been found to modify the AMPA receptor GluR2 and G-protein coupling receptors, i.e, SR2C and GABA receptor. The knockout animal models (Wang lab) have shown that this molecule is essential for embryonic survival and tissue homeostasis. However the molecular mechanism is largely unknown. The Bahar lab has initiated a new collaboration with the Wang Lab, to help investigate (a) the effects of the mutations induced upon ADAR1 editing on calcium channeling properties of iGluRs, (ii the effect of specific mutations on the functions of serotonin receptors and potassium channels, using molecular modeling, and (c) mathematical modeling and quantitative assessment of the effect of suppressing cytosolic RNA pathway by ADAR1 on relevant cellular pathways networks.

In progress


 

19) Modeling Dendritic Sparks at Dendritic Branch Points

PI: Terry Sejnowski and Tom Bartol, Salk Institute  Collaborators: Mark Ellisman, University of California at San Diego

The purpose of this C&SP is to collaborate with Mark Ellisman's lab to create realistic MCell models of calcium sparks that occur at dendritic branch points.

Neuronal calcium events similar to calcium sparks in cardiac myocytes have been observed to occur at dendritic branch points (Manita and Ross, J Neurosci, 2009). The origin of these events has been attributed to ryanodine receptors (RyRs) and IP3 receptors (IP3Rs). The Ellisman lab is working to reconstruct the subcellular ultrastructure of dendritic branch points and to identify the localization and relative concentrations of channels and molecules involved in calcium signaling and processing, including: ryanodine receptors (RyR1, RyR2, RyR3), IP3R), metabotropic glutamate receptors (mGluR), various voltage-gated calcium channels (T-Type, L-Type, and N-Type), various calcium-activated potassium channels (BK, SK, Kv4.2), and sodium channels. From these data we will help the Ellisman lab create a realistic model of dendritic branch points that incorporates both voltage dynamics at the neuronal level and calcium and other molecular dynamics at single branch points with via a combined NEURON and MCell system where both are using realistic morphologies from the same sample.

In progress


 

29)  Spatiotemporal models of autophagy

PI: Joern Dengjel, University of Frieberg Collaborator: R. Murphy, Carnegie Mellon University

Macroautophagy is thought to be mainly cytoprotective, although it also has been linked to autophagic cell death (Feng et al., 2015). Autophagosomes are de-novo formed double membrane vesicles, which enwrap cytoplasm and target it for lysosomal degradation. Their biogenesis is complex and still holds many open questions (Lamb et al., 2013). We will generate wide-field as well as confocal fluorescent images of forming autophagosomes using different stress conditions such as amino acid and growth factor starvation. We will use cells stably expressing the autophagosomal marker MAP1LC3B (LC3) coupled to mRFP-GFP which allows the analysis of autophagosome turnover (Kimura et al., 2007). Also, we will generate images of all six fluorescently tagged isoforms of LC3 to determine if different isoforms have an influence on site of formation of autophagosomes and size/dynamics of vesicles. We will use these to construct image-derived models to enable comparison of different conditions.

Literature Cited

    • Feng, Y., Z. Yao, and D.J. Klionsky. (2015) How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy. Trends Cell Biol.25:354-363.
    • Kimura, S., T. Noda, and T. Yoshimori. (2007) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 3:452-460.
    • Lamb, C.A., T. Yoshimori, and S.A. Tooze. 2013. The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol.14:759-774.

Publications Resulting from This Work

    • C. Gretzmeier, S. Eiselein, G. R. Johnson, R. Engelke, H. Nowag, M. Zarei, V. Küttner, A. C. Becker, K. T. G. Rigbolt, M. Høyer-Hansen, J. S. Andersen, C. Münz, R. F. Murphy, and J. Dengjel (2017) Degradation of protein translation machinery by amino acid starvation-induced macroautophagy. Autophagy, in press.

In progress