Gina Sosinsky - US grants

The gated connexon channels of gap junction membranes regulate the intercellular communication essential for differentiation and other integrated tissue activities. The cytoplasmic portion of the connexon appears to be flexible, while the transmembrane domain has a relatively invariant structure. Gap junctions are composed of a family of related proteins called connexins which can localize in the same junctional plaque. Determination of the sequences of the connexin proteins and correlation of information on antibody binding, proteolytic cleavage and chemical reactivity have led to a model where the C- and N-termini are located at the cytoplasmic surface and four alpha-helical segments of the protein cross the membrane. Objectives of this proposal are: 1.To obtain electron micrographs of complete and split gap junctions either with antibody or chemical labels or subjected to partial proteolysis. The positions of these labels/protein modifications will be correlated with the amino acid sequence to develop an improved model of gap junction structure. 2.To use the Scanning Transmission Electron Microscope at Brookhaven to record dark field micrographs of gap junction connexon arrays in order to determine the mass of the connexon and the distribution of Connexin32 and Connexin26 in connexons. 3.To obtain a low-resolution three-dimensional reconstruction of the flexible cytoplasmic domains from thin section data. 4.To continue effort to develop better-ordered specimens and improve embedding media for high resolution (> 10 alpha) electron microscopic structure determination.

While the major focus of activity at the NCMIR involves facilitation of investigations into cellular structure and function through coordinated application of light and electron microscopy techniques, part of NCMIR's mission is to provide a regional resource for molecular microscopists as well. In order to service these investigators, the JEOL 4000 is available for low temperature/low irradation imaging. In the past year, there has not been any usage of the JEOL 4000 for these applications because our previous users have either used the JEOL 4000 for tomographic projects (Frey, Sosinsky) or have acquired a suitable microscope in their own laboratories (Yeager, Milligan).

Electron microscope tomographic reconstruction has proven to be an invaluable technique for studying the three structure (Perkins et al., 1997a and b). This project area includes four specific projects: 1) development of methods for processing large tomographic data at remote facilities; 2) implementing procedures that enable acquisition and processing of data from multiple tilt axes to improve the z axis resolution; 3) examining tomographic reconstructions of computer generated models; and working on the development of procedures for serial tomography. During the last year we have focused on the following: Double axis tilt reconstruction: The Z resolution in electron tomography can be significantly improved by employing a double axis tilt procedure. In this acquisition method, images are acquired by tilting on the y axis and then rotating the specimen 908 with a rotating specimen holder and performing a second tilt series acquisition. With a 160 tilt series, the loss of frequencies is reduced from 33% (single axis) to 16.1%, thereby increasing the Z resolution. In fact, this value is very close to that obtained using the more image intensive and difficult conical tilting procedure (13.4% missing region). We are implementing two different procedures for deriving reconstructions from double-tilt data that have recently been reported. We have the obtained programs from David Mastronarde at the Boulder NCRR funded Electron Microscope Resource and are experimenting with his procedure and integrating it into our tomographic processing stream. We are also developing software compatible with a our tomography programs to perform another double technique from the Albany resource. We plan to evaluate both these methods for double tilt tomography on models and data specimens generated at NCMIR. Methods for improved tomographic reconstruction and processing large tomographic datasets at remote facilities: Although, we have generally employed the R-weighted backprojection algorithm to reconstruct the 3D volume from the tilt-series projections, we have begun investigating the application of iterative techniques such as ART or SIRT following the R-weighted method to improve the reconstruction, using programs written at NCMIR. The application of the combination of the R-weighted and iterative methods to large reconstructions is computationally intensive. In order to expedite processing we have implemented the R-weighted, ART and SIRT reconstruction algorithms on the 400-node Intel Paragon, and more recently, on the 256-processor Cray T3E parallel supercomputer at SDSC. This parallel implementation was partially supported by the NCRR funded National Biological Computing Resource (NBCR) at SDSC. These single-axis tilt reconstruction algorithms are relatively straight forward to implement on massively parallel supercomputers. The reconstruction of each of the one voxel-thick planes orthogonal to the tilt axis of the volume is assigned to an individual node on the parallel computer. Using a small number of nodes (16-32) to minimize queuing delays, the parallel implementation is ten times faster than that of a high-performance, single-processor machine such as the Silicon Graphics Incorporated workstation with an R10000 processor. A script, written in PERL, enables the researcher to define processing parameters easily and to initiate the reconstruction remotely on the parallel computer, thereby bypassing the complexities of the interface to the parallel machines. During this last year the parallel reconstruction program has been used by investigators in collaborative projects examining 1) changes in the structure of dendritic spines following loss of synaptic input, alterations in the structure of cardiac muscle in an animal model of heart failure, and 2) an analysis of the complex three-dimensional structure of mitochondria, 3) synaptic transmission in cultured neurons and frog-hair cell receptors. In addition to facilitating research projects using tomography, the increased speed of computation afforded by the use of parallel supercomputers will be useful in comparing the relative merits of various reconstruction methods and in determining the optimal parameters for a given reconstruction algorithm, e.g. the number of iterations (see below). The design of the parallel tomography program is modular. therefore, it is relatively straight forward to incorporate more recent single-axis, tilt-reconstruction algorithms that may further improve the quality of tomographic reconstructions. We are currently implementing a method developed by Jose Carazo and colleagues using spherical basis functions ("blobs") which is reported to decrease artifacts and improve the resolution of features within the reconstruction. Improved autotomography: We are now implementing a low-dose autotomography data acquisition system from Hans Tietz. We have implemented an interface to the IVEM microscope computer and computerized stage. Over the next year, we plan to adapt this control package to accommodate the larger image format of the 2k x 2k camera system (described above in the section on development of the slow scan camera and interface).

While the major focus of activity at the NCMIR involves facilitation of investigations into cellular structure and function through coordinated application of light and electron microscopy techniques, part of NCMIR's mission is to provide a regional resource for molecular microscopists as well. In order to service these investigators, we have set up software for molecular microscopy. Packages for molecular microscopy that were previously installed include the MRC 2D crystallographic analysis package, ICE, SPECTRA, CCP4, SUPRIM and PHOELIX. These packages are and will be maintained and updated. During the past year, we have investigated various programs or suite of programs for fitting of independent structures obtained by structural techniques with differing levels of resolution (e.g. electron microscopy, light microscopy, X-ray crystallography). A long-term goal of NCMIR is the development or application of programs for integrating data obtained from structural biology techniques (molecular microscopy, confocal microscopy, EM tomography, X-ray crystallography). Programs we have tried with the gap junction EM data include: Xtalview, O, Neural Editor and Ducky. In particular, we found that Xtalview, a package developed for X-ray crystallographic analysis, suited the needs of fitting independent gap junction data sets (see Perkins et al., J. Mol. Biol. 272, 1998). For this purpose, Mr. Lamont wrote a program for converting the SYNU surface representations (our visualization software) into protein database coordinates (PDB files) as well as a program that then takes modified pdb file and converts it back to SYNU representations. These pseudo-PDB files can then inputed into any programs that display protein coordinates (Xtalview, O, Rasmol). We also tried using Xtalview to fit a confocal and EM tomographic data set. This worked with limited success. In the next year, we will also investigate AVS, which may have more flexibility than the current X-ray crystallographic software, as a fitting and visualization environment. In conjunction with the San Diego Supercomputer Center (SDSC) and with supplemental support by the National Science Foundation, Dr. Sosinsky created the EM Outreach Program. The goals of this program are twofold. First, to develop NCMIR as a software environment and resource for tools and expertise for research and second, to provide educational materials as well as research tools via web based technologies. In order to take advantage of the national infrastructure afforded to us by SDSC, the EM Outreach program is located on an SDSC Web Site. The EM Outreach program is found at the URL: em-outreach.sdsc.edu.. The EM Outreach program contains (1) information in the form of Web pages about EM software applications or documentation and (2) a course complete with text and figures in the form of Web documents on structure analysis for electron microscopy (original source material for the web course was obtained from Dr. Tim Baker, Purdue University). The EM Outreach program ha s been advertised at the 1997 Gordon Conference on 3D Microscopy, Microscopy Society of America 1997 meeting and the 1997 Neuroscience meeting. Our plans for the coming year include expanding the software pages as well as revising the text and figures in current educational web pages, creating up/down Links within texts, incorporating a search engine for topics (index), links to appropriate references, links to appropriate software in image processing section, links to tutorials on how to use software and implementing any animations and/or advanced web technology.

In metazoans, individual cells are functionally differentiated and organized into tissues and organs whose separate functions collectively support the existence of the whole organism. Within tissues and organs, proper functionality generally requires communication between cells in order to coordinate cellular activities, and some of this communication occurs via direct exchange of small molecules between neighboring cells through intercellular channels known as gap junction channels. These intercellular channels are critical for integrating and regulating basic cell processes such as metabolic cooperation, ionic transmission, differentiation and functional regulation.

The best studied of these are the gated vertebrate gap junctional channels which are comprised of connexin. The channels consist of a dimer of two hexamers (connexon) of the constituent protein (connexin). These channels are unique in that when the two hexamers pair up across two neighboring cells, they self-assemble into characteristic cellular junctional areas (plaques) that are morphologically distinctive. The connexins are a family of related proteins sharing a common folding motif. Different connexins are found in the same tissue and different connexins can be localized to the same gap junctional plaques. In the current structural model, the C and N termini are located on the cytoplasmic side of the membrane and cross the membrane four times in alpha-helical segments. Mutagenesis studies have indicated that the extracellular domains may contain beta sheet structures.

Non-connexin gap junction channels have been discovered experimentally in invertebrates, and the protein components of these invertebrate gap junction channels (called innexins) have recently been cloned; these have no sequence homology with the vertebrate connexins. Subsequently, the availability of human and mouse genomic information and bioinformatics tools and approaches have resulted in the identification of three mammalian homologues to innexins. Additional homologues of innexins have also been found in amphibia. This new class of vertebrate homologues of innexins has been termed the pannexins, and it has been hypothesized that the pannexins have a similar membrane topology as connexins despite their lack of sequence homology. In addition, the physiological attributes of innexins and pannexins (large single channel conductances, ability to readily make hemichannels as well as intercellular channels in Xenopus oocytes, lack of a Ca2+ induced gating response) set them apart from connexin channels. However, almost nothing is known about the domains they form within cells, expression in tissues at the protein level, their molecular structure or even if they do make gap junction-like structures in vivo.

The Sosinsky laboratory is focusing on the structure and expression of two pannexins, pannexin1 (Px1) and pannexin2 (Px2), using methods for light and electron microscopy that Dr. Soskinsky has developed and expanded in her laboratory and previously applied to connexin-based gap junctions. There are two specific aims for this research project:
1. To express and monitor tagged Px1 and Px2 in tissue culture cells for correlative light and electron microscopy, to address whether Px1 and Px2 channels form gap junction-like structures and if so, to determine their shape and dimensions. Two approaches will be used: Quantum dot labeling of standard small peptide additions to the sequence (e.g. myc, HA) and fluorescence photooxidation of the ReAsH bound to the tetracysteine domain of Pannexin-Green Fluorescent Protein (GFP)-tetracysteine or Px-tetracysteine chimeras to allow for identification of these structures at EM resolution. Since preliminary work has shown that tagged versions of these constructs do not traffic exactly the same as wild-type, Dr. Sosinsky will also develop and characterize antibodies to these proteins for use as probes for the analysis expression patterns in stable cultured mammalian cell lines and in native mammalian tissues.
2. To purify and characterize Px1 and Px2 channels and hemichannels isolated from stably expressing mammalian cell lines and baculovirus expressed Sf9 cells, to address whether pannexin channels contain the same symmetry and oligomerization number as connexin channels, as has been hypothesized from electron micrographs of innexin structures.

INTELLECTUAL MERIT: The overall goal is to characterize this recently discovered and novel set of membrane proteins in vitro and in vivo and determine if pannexins do form the basis for a hitherto-unknown avenue of intercellular communication in vertebrates. Connexin-based ap junctions are found in essentially all tissues of vertebrate body plans. Thus, there is great interest in understanding what role these newly discovered, putative gap junctions or hemichannels would play in complementing connexin-based intercellular channel communication in vertebrates. It may well be that current textbook ideas on vertebrate gap junctions would need revising pending the outcome of these studies.

BROADER IMPACT: Dr. Sokinsky's technical developments and achievements in correlative microscopies are already being disseminated in conferences and classrooms. This work will provide training opportunities for postdoctoral and undergraduate students.

The purpose of this grant request is to obtain equipment for adapting our high resolution JEOL 2000 electron microscopy for cryo-imaging and diffraction. The requested equipment includes cold stages, an anticontaminator, a TV-rate camera, slow-scan CCD camera and accessory equipment for maintenance of the cold stages and anticontaminator. Our research programs in structural biology involve computerized image analysis of electron micrographs, coordinated with X-ray crystallography, biochemical and biophysical techniques. To make full use of these capabilities, it is essential that we upgrade our electron microscope facilities. At present, there is no microscope on the University of California, San Diego campus that is dedicated to cryo-microscopy. The only microscope on the campus that has a cold stage and anticontaminator is the IVEM JEOL 4000. This microscope is dedicated to electron tomography and is overloaded with users. In contrast, our JEOL 2000 electron microscope experiences less use and this microscope would be extremely suitable for cryo-imaging. The eight major programs requiring low temperature data are: (1) Molecular Structure of Gap Junction Membrane Channels (Sosinsky); (2) Cryomicroscopy of PKA-DAKAP Complexes (Taylor); (3) Cryomicroscopy of PKA-Decorated Cowpea Mosaic Virus (Johnson); (4) Structural Studies of Cellular Components in the Node of Ranvier (Ellisman); (5) Structural Investigation of Bacterial Pathogen Proteins (P. Ghosh); (6) Structural Study of an Enhanceosome (G.Ghosh); (7) Visualization of Recombination Intermediates by Cryo-EM (Segall); (8) Structural Investigations of Conformational Variations Due to Cation or Ligand Binding in Integrins (Stuiver). These eight research projects involve 23 investigators that would depend heavily on the use of a cryo-microscope. In each of these research projects, the primary goal is to determine the three-dimensional structure of organized macromolecular systems and to understand their functions in terms of the dynamic state of their structures. A low-temperature stage would allow samples to be examined in an unstained frozen-hydrated state or if stained, would take advantage of the increased radiation resistance due to the liquid nitrogen temperature. A fully equipped JEOL 2000 would have the full range of these capabilities required for progress in our research programs in structural biology.

DESCRIPTION (provided by applicant): The purpose of this grant request is to obtain funds to purchase and install a High Pressure Freezer (HPF) machine. This unit will be used to prepare well-preserved specimens for tomography applications. High pressure freezing delivers the best specimen preparation method for preserving cell and tissue ultrastructure and therefore, obtaining high resolution in situ data. The requested equipment is a Bal-Tec 11PM 010 High Pressure Freezing Machine (HFP) and accessories for sample preparation. Our research programs in structural biology involve the three-dimensional reconstruction of cells and tissues using thick sections and intermediate voltage electron microscopy (IVEM). We coordinate and integrate our structural studies with data obtained from molecular microscopy, X-ray crystallography, biochemical, biophysical and neurophysiological techniques. Our goal is to accurately reconstruct the structure of cells and organelles using the best preparative, microscopy and computational methods. At present, the closest HFP units are located in the San Francisco Bay Area or the Tucson, Arizona. The HFP will be housed in the National Center for Microscopy and Imaging Research at the University of California, San Diego. The five major programs requiring the use of the HFP are: (1) Three-dimensional cellular organization of gap junction membrane channels (Sosinsky, P.!.); (2) Structural studies of cellular components in the Node of Ranvier (Ellisman, P.1.); (3) Changes in macromolecular associations in cerebellar Purkinje neuron dendritic spines during synaptic-depression (Tsien and Lev-Ram, P.1?s); (4) Mechanisms of non-enveloped virus entry (Johnson and Schneemann, P.I.?s); and (5) Three-dimensional structure and function of mitochondria as viewed by electron tomography (Frey, P.1.). In addition, four collaborative projects from the NCMIR Research Resource that would immediately benefit from the usage of the HFP are described briefly. These nine research projects involve over 25 investigators that would depend heavily on the use of the HPF. In all of these research projects, the primary goal is to understand the three-dimensional structure of organized cellular components and relate this morphology to their functionality.

DESCRIPTION (provided by applicant): Gap junctions serve an essential role in the passage of molecules from the cytoplasm of one cell to its neighbor in both functional and homeostatic capabilities. They are defined as clusters of closely packed intercellular membrane channels embedded in the plasma membranes of two adjoining cells. The channels are composed of two hexamers of a protein (connexon) from a family of integral membrane proteins known as connexins. Here, we focus on the structure and function of connexin26 (Cx26), the smallest of the family. Mutations in the DNA sequence can result in hereditary sensorineural deafness and account for between one third to one half of the cases of prelingual inherited deafness in Caucasian populations. We have isolated preparations of Cx26 gap junctions in pure and sufficient amounts for biochemical and structural studies. These 2D crystals are amenable to electron microscopy (EM) structure determination and conformational dynamics as revealed with atomic force microscopy (AFM) done under hydrated conditions. In SPECIFIC AIM 1, we will determine the structure of the Cx26 hemichannel beyond 10 Angstroms using state of the art cryo-EM and improvements on image processing procedures. This involves improving specimen preparation, imaging at either liquid nitrogen or liquid helium temperature and implementation of a combined single particle/ 2D crystallographic approach to circumvent imperfect crystal lattices. In SPECIFIC AIM 2, we will construct Cx26 wild type and mutant cell lines with a tetracysteine domain genetic tag to improve isolation with FlAsH ligand affinity bead purification, stably express these in HeLa cells or in baculovirus-infected Sf9 insect cells and isolate the gap junctions or connexons for structural analysis using the methods developed in Specific Aim 1. We will construct two Cx26 mutants (P97L and T135A), each containing a single point mutation in one of the transmembrane helices that changes the effective pore properties. These mutations should be reflected in conformational changes in the 3D structure. In SPECIFIC AIM 3, we will expand coordinated AFM/EM experiments for visualizing conformational changes due to treatments known to close or alter gap junction mediated communication. Preliminary AFM images have visualized conformational changes at submolecular resolution. We have chosen five treatments that known to induce closure of Cx26 channels or hemichannels and are physiologically relevant. Conformational changes identified by AFM imaging will be further imaged using EM. Each of these goals is intended to complement the others and lead to structural and physiological models of Cx26 germane to the entire connexin family.

Abstract not provided.

Abstract not provided.

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DESCRIPTION (provided by applicant): Gap junctions (GJ) are defined as clusters of closely packed membrane channels containing the connexin protein, connecting two adjoining cells. The channel is composed of two hexamers from each cell and contains oligomers of one or more connexins. GJ serve important functions in direct intercellular communication in almost all vertebrates cell types. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Normal cell and tissue homeostasis as well as developmental processes are dependent on the proper trafficking of connexins and the fast turnover rate of connexins has been hypothesized to be one mechanism of channel function regulation. Many of the mutations in connexin diseases (sensorineural deafness, Charcot-Marie-Tooth disease, cataracts, for example) result in abnormal trafficking. This proposal focuses on the identification and characterization of connexin trafficking structures using the techniques of tetracysteine genetic tags complexed with biarsenical fluorescent ligands, optical pulse-chase, fluorescence photooxidation, correlative light and electron microscopy and electron tomography to produce 3D reconstructions of selectively labeled connexins in cells. Using these techniques in combination, we aim to study these intermediates at reasonably high electron tomographic resolution (approximately 40-60 Angstroms) in 3D to determine their composition and locations within the context of other cellular components. We have four specific aims for the requested five-year period. Specific Aim 1 follows up on our initial study of Gaietta et al., (2002) by dissecting "unstimulated" or normal connexin trafficking pathways in endogenously expressing connexin cell lines. Initial studies were done in HeLa cells, a cell line known to produce artificially large quantities of connexins. We are expanding our initial study to investigate cell lines that are unpolarized, polarized and primary to see if the mechanism of adding new gap junction channels to the edges of the plaques is a universal one. Specific Aim 2 explores the question of whether hemichannels are a stable part of the plasma membrane or a short-lived trafficking intermediate. Specific Aim 3 is focused on the connexin structures found during mitosis and whether recycling of connexins occurs. Specific Aim 4 investigates investigate the role that cholesterol plays in plaque maintenance by examining the effect of cholesterol depleting agents and the re-uptake of cholesterol temporally examining this process at higher resolution and in 3-D than previously published.

This award will provide support for qualified undergraduates, graduate students, and postdoctoral researchers to attend the International Congress on Electron Tomography (ICET) in San Diego, CA, from November 5-8, 2006. This congress is the fourth in a series of workshops that was initiated in 1997 to begin to bring together practitioners developing and applying electron microscopic tomography. The meetings have provided a forum for intensive interactions and have catalyzed technical developments and an increased number of users. At this juncture, electron tomography is one of the most rapidly evolving approaches in multiscale 3D microscopy and is being employed for an increasingly broad range of applications, well beyond those envisioned at the time of the first conference.

Electron Tomography is a new research area that produces 3-dimensional quantitative images from electron microscopy. Electron tomography is moving from a specialized experimental technique practiced by a few laboratories to one that is delivering critical new information to cell biologists, structural biologists and neuroscientists. Because electron tomography has been identified as a growth field, it is anticipated that the graduate students and postdoctoral researchers being trained today will go on to become the next generation lead investigators.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. SPECIMEN PREPARATION, PRESERVATION AND STAINING TECHNOLOGIES FOR MAPPING PROTEINS, CELLULAR COMPLEXES, PATHWAYS AND THE SUPRAMOLECULAR ORGANIZATION OF TISSUES USING MULTIPLE LABELS AND MULTISCALE IMAGING. Our efforts in this area focused on continuing to develop technologies to enable imaging of proteins and macromolecular complexes using multiple labels and multiscale microscopies. These approaches include (1) expanding our repertoire of fluorescent compounds used for multiscale imaging;(2) combining high-pressure freezing/freeze-substitution with molecular labeling approaches to further enhance preservation of ultrastructure;(3) adapting metal chelating derivatives of diaminobenzidine to enable simultaneous multi-protein EM labeling using electron energy loss spectroscopy (EELS), and other detection methods;(4) and exploring the feasibility of cathodoluminescence (CL) imaging using quantum dots, nanophosphors and fluorescent proteins.